Last updated on March 16th, 2023 at 12:09 am
Title 40 – Protection of Environment–Volume 35
CHAPTER I – ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
SUBCHAPTER R – TOXIC SUBSTANCES CONTROL ACT (CONTINUED)
PART 790 – PROCEDURES GOVERNING TESTING CONSENT AGREEMENTS AND TEST RULES
Subpart A – General Provisions
(a) This part establishes procedures for gathering information, conducting negotiations, and developing and implementing test rules or consent agreements on chemical substances and mixtures under section 4 of TSCA.
(b) Section 4 of the Act authorizes EPA to require manufacturers and processors of chemical substances and mixtures to test these chemicals to determine whether they have adverse health or environmental effects. Section 4 (a) empowers the Agency to promulgate rules which require such testing. In addition, EPA has implied authority to enter into enforceable consent agreements requiring testing where they provide procedural safeguards equivalent to those that apply where testing is conducted by rule.
(c) EPA intends to use enforceable consent agreements to accomplish testing where a consensus exists among EPA, affected manufacturers and/or processors, and interested members of the public concerning the need for and scope of testing.
§ 790.2 Applicability.
This part is applicable to manufacturers and processors of chemical substances or mixtures who are subject to the testing requirements of a consent agreement or a rule under section 4(a) of the Act. The procedures for test rules are applicable to each test rule in part 799 or this chapter unless otherwise stated in specific test rules in part 799 of this chapter.
§ 790.3 Definitions.
Terms defined in the Act and not explicitly defined herein are used with the meaning given in the Act. For the purpose of this part:
Act means the Toxic Substances Control Act, 15 U.S.C. 2601 et seq.
Additive means a chemical substance that is intentionally added to another chemical substance to improve its stability or impart some other desirable quality.
Central Data Exchange or CDX means EPA’s centralized electronic submission receiving system.
Chemical means a chemical substance or mixture.
Chemical Information Submission System or CISS means EPA’s electronic, web-based tool for the completion and submission of data, reports, and other information, or its successors.
Consortium means an association of manufacturers and/or processors who have made an agreement to jointly sponsor testing.
EPA means the U.S. Environmental Protection Agency.
Equivalence data means chemical data or biological test data intended to show that two substances or mixtures are equivalent.
Equivalent means that a chemical substance or mixture is able to represent or substitute for another in a test or series of tests, and that the data from one substance can be used to make scientific and regulatory decisions concerning the other substance.
Exemption means an exemption from a testing requirement of a test rule promulgated under section 4 of the Act and part 799 of this chapter.
Impurity means a chemical substance which is uninitentionally present with another chemical substance.
Joint sponsor means a person who sponsors testing pursuant to section 4(b)(3)(A) of the Act.
Joint sponsorship means the sponsorship of testing by two or more persons in accordance with section
Person means an individual, partnership, corporation, association, scientific or academic establishment, or organizational unit thereof, and any other legal entity.
Principal sponsor means an individual sponsor or the joint sponsor who assumes primary responsibility for the direction of a study and for oral and written communication with EPA.
Protocol means the plan and procedures which are to be followed in conducting a test.
Reimbursement period refers to a period that begins when the data from the last non-duplicative test to be completed under a test rule are submitted to EPA and ends after an amount of time equal to that which had been required to develop data or after five years, whichever is later.
Sponsor means the person or persons who design, direct and finance the testing of a substance or mixture.
Test substance means the form of chemical substance or mixture that is specified for use in testing.
§ 790.5 Submission of information.
(a) All submissions and correspondence to EPA under this part must bear the Code of Federal Regulations (CFR) section number of the subject chemical test rule consent agreements.
(b) You must use the CISS tool to complete and submit via CDX all data, reports, other information, and correspondence required by rules promulgated under TSCA section 4, and for correspondence pertaining to consent agreements as required under this part. The submissions must be made only as set forth in this section.
(c) To access the CISS tool go to https://cdx.epa.gov/ssl/CSPP/PrimaryAuthorizedOfficial/Home.aspx and follow the appropriate links and for further instructions to go http://www.epa.gov/oppt/chemtest/ereporting/index.html.
§ 790.7 Confidentiality.
(a) Any person subject to the requirements of a consent agreement or a test rule under section 4 of the Act may assert a claim of confidentiality for certain information submitted to EPA in response to the consent agreement or the test rule. Any information claimed as confidential will be treated in accordance with the procedures in part 2 of this title and section 14 of the Act. Failure to assert a claim of confidentiality at the time the information is submitted will result in the information being made available to the public without further notice to the submitter.
(b) A claim of confidentiality must be asserted by circling or otherwise marking the specific information claimed as confidential and designating it with the words “confidential business information,” “trade secret,” or another appropriate phrase indicating its confidential character.
(c) If a person asserts a claim of confidentiality for study plan information described in §§ 790.50(c)(1)(iii)(D), (iv), (v), and (vi) and 790.62(b)(6), (7), (8), (9), and (10), the person must provide a detailed written substantiation of the claim by answering the questions in this paragraph. Failure to provide written substantiation at the time the study plan information is submitted will be considered a waiver of the claim of confidentiality, and the study plan information will be disclosed to the public without further notice.
(1) Would disclosure of the study plan information disclose processes used in the manufacture or processing of a chemical substance or mixture? Describe how this would occur.
(2) Would disclosure of the study plan information disclose the portion of a mixture comprised by any of the substances in the mixture? Describe how this would occur.
(3) What harmful effects to your competitive position, if any, do you think would result from disclosure of this information? How would a competitor use such information? How substantial would the harmful effects be? What is the causal relationship between disclosure and the harmful effects?
(4) For what period of time should confidential treatment be given? Until a specific date, the occurrence of a specific event, or permanently? Why?
(5) What measures have you taken to guard against disclosure of this information to others?
(6) To what extent has this information been disclosed to others? What precautions have been taken in connection with such disclosures?
(7) Has this information been disclosed to the public in any forms? Describe the circumstances.
(8) Has the information been disclosed in a patent?
(9) Has EPA, another Federal agency, or any Federal court made any pertinent confidentiality determination regarding this information? If so, copies of such determinations must be included in the substantiation.
(d) If the substantiation provided under paragraph (c) of this section contains information which the submitter considers confidential, the submitter must assert a separate claim of confidentiality for that information at the time of submission in accordance with paragraph (b) of this section.
Subpart B – Procedures for Developing Consent Agreements and Test Rules
§ 790.20 Recommendation, recommendation with an intent to designate, and designation of testing candidates by the ITC.
(a) ITC recommendations and recommendations with intent to designate. The ITC has advised EPA that it will discharge its responsibilities under section 4(e) of TSCA in the following manner:
(1) When the ITC identifies a chemical substance or mixture that it believes should receive expedited consideration by EPA for testing, the ITC may add the substance or mixture to its list of chemicals recommended for testing and include a statement that the ITC intends to designate the substance or mixture for action by EPA in accordance with section 4(e)(1)(B) of TSCA.
(2) Chemical substances or mixtures selected for expedited review under paragraph (a)(1) of this section may, at a later time, be designated for EPA action within 12 months of such designation. The ITC’s subsequent decision would be based on the ITC’s review of TSCA sections 8(a) and 8(d) data and other relevant information.
(3) Where the ITC concludes that a chemical substance or mixture warrants testing consideration but that expedited EPA review of testing needs is not justified, the ITC will add the substance or mixture to its list of testing recommendations without expressing an intent to designate the substance or mixture for EPA action in accordance with section 4(e)(1)(B) of TSCA.
(4) The ITC reserves its right to designate any chemical substance or mixture that it determines the Agency should, within 12 months of the date first designated, initiate a proceeding under section 4(a) of TSCA.
(b) Preliminary EPA evaluation of ITC recommendations with intent to designate. Following receipt of an ITC report containing a recommendation with an intent to designate, EPA will use the following procedure for completing a preliminary evaluation of testing needs on those chemical substances that the ITC has recommended with intent to designate:
(1) EPA will publish the ITC report in the
(2) EPA will publish a
(3) EPA will hold a public “focus meeting” to discuss the ITC’s testing recommendations and obtain comments and information from interested parties.
(4) EPA will evaluate submissions received under TSCA sections 8(a) and 8(d) reporting requirements, comments filed on the ITC’s recommendations, and other information and data compiled by the Agency.
(5) EPA will make a preliminary staff determination of the need for testing and, where testing appears warranted, will tentatively select the studies to be performed.
(6) EPA will hold a public meeting to announce its preliminary testing determinations.
(c) EPA response to ITC designations and recommendations – (1) Where a chemical substance or mixture is designated for EPA action under section 4(e)(1)(B) of TSCA, the Agency will take either one of the following actions within 12 months after receiving the ITC designation:
(i) Initiate rulemaking proceedings under section 4(a) of TSCA. Where the testing recommendations of the ITC raise unusually complex and novel issues that require additional Agency review and opportunity for public comment, the Agency may initiate rulemaking by publishing an Advance Notice of Proposed Rulemaking (ANPRM).
(ii) Publish a
(2) Where a chemical substance or mixture has been recommended for testing by the ITC, whether with or without an intent to designate, EPA will use its best efforts to act on the ITC’s recommendations as rapidly as possible consistent with its other priorities and responsibilities. EPA may respond to the ITC’s recommendations with action such as:
(i) Initiating rulemaking proceedings under section 4(a) of TSCA,
(ii) Publishing a
(iii) Entering into a consent agreement in accordance with this subpart.
§ 790.22 Procedures for developing consent agreements.
(a) Preliminary EPA evaluation of proposed consent agreement. Where EPA believes that testing of a chemical substance or mixture may be needed, and wishes to explore whether a consent agreement may satisfy the identified testing needs, EPA will invite manufacturers and/or processors of the affected chemical substance or mixture to submit a proposed consent agreement to EPA. EPA will evaluate the proposal(s) and may request additional clarifications of or revisions to the proposal(s).
(b) Negotiation procedures for consent agreements. If, after evaluating the proposed consent agreement(s), EPA believes it is likely that proceeding with negotiation of a consent agreement would be an efficient means of developing the data, EPA will use the following procedures to conduct such negotiations:
(1) In the
(2) The Agency will meet with interested parties at the negotiation meeting(s) for the purpose of attempting to negotiate a consent agreement. Only the submitter(s) of the proposal(s) that is the basis for negotiation and those persons who submit written requests to participate in or monitor negotiations by the deadline established under paragraph (b)(1) of this section will be deemed “interested parties” for purposes of this section.
(3) All negotiation meetings will be open to members of the public, but only interested parties will be permitted to participate in negotiations. The minutes of each meeting will be prepared by EPA. Meeting minutes, the proposed consent agreement(s), background documents, and other materials distributed at negotiation meetings will be placed in an Internet-accessible public docket established by EPA.
(4) If EPA concludes at any time that negotiations are unlikely to produce a final agreement, EPA will terminate negotiations and may proceed with rulemaking. If EPA terminates negotiations, no further opportunity for negotiations will be provided. EPA will notify all interested parties of the termination.
(5) The period between the first negotiation meeting and final agreement, if any (“the negotiation period”), will be no longer than 6 months, unless extended prior to its expiration in accordance with paragraph (b)(7) of this section. This period will include all negotiation meetings, and the processes discussed in paragraphs (b)(6) and (b)(9) of this section. If the negotiation period passes without the production of a final agreement, negotiations and development of the subject ECA will terminate automatically.
(6) EPA will circulate a draft of the consent agreement to all interested parties if EPA concludes that such draft is likely to achieve final agreement. A period of 30 days will be provided for submitting comments or written objections under paragraph (b)(8)(i)(B) of this section.
(7) If, prior to the expiration of the negotiation period, final agreement has not been reached, EPA may at its discretion provide one or more extensions, each of which may be up to 60 days, if it seems likely to EPA that a final agreement will be reached during that time. EPA will notify all interested parties of any extension(s).
(8) (i) EPA will enter into consent agreements only where there is a consensus among the Agency, one or more manufacturers and/or processors who agree to conduct or sponsor the testing, and all other interested parties who identify themselves in accordance with paragraph (b)(2) of this section. EPA will not enter into a consent agreement in either of the following circumstances:
(A) EPA and affected manufacturers and/or processors cannot reach a consensus in the timeframe described in paragraph (b)(5) of this section.
(B) A draft consent agreement is considered inadequate by other interested parties who have submitted timely written objections to the draft consent agreement, which provide a specific explanation of the grounds on which the draft agreement is objectionable.
(ii) EPA may reject objections described in paragraph (b)(8)(i)(B) of this section only where the Agency concludes the objections:
(A) Are not made in good faith;
(B) Are untimely;
(C) Do not involve the adequacy of the proposed testing program or other features of the agreement that may affect EPA’s ability to fulfill the goals and purposes of TSCA; or
(D) Are not accompanied by a specific explanation of the grounds on which the draft agreement is considered objectionable.
(iii) The unwillingness of some manufacturers and/or processors to sign the draft consent agreement does not, in itself, establish a lack of consensus if EPA concludes that those manufacturers and/or processors who are prepared to sign the agreement are capable of accomplishing the testing to be required and that the draft agreement will achieve the purposes of TSCA in all other respects.
(9) Where a consensus exists, as described in paragraph (b)(8) of this section, concerning the contents of a draft consent agreement, the draft consent agreement will be circulated to EPA management and the parties that are to conduct or sponsor testing under the agreement, for final approval and signature.
(10) Upon final approval and signature of a consent agreement, EPA will publish a
Subpart C – Implementation, Enforcement, and Modification of Test Rules
§ 790.40 Promulgation of test rules.
(a) If EPA determines that it is necessary to test a chemical substance or mixture by rule under section 4 of the Act, it will promulgate a test rule in part 799 of this chapter.
(b) EPA will promulgate specific test rules in part 799 of this chapter either by a single-phase rulemaking procedure or by a two-phase rulemaking procedure.
(1) Under single-phase test rule development, EPA will promulgate a test rule in part 799 of this chapter through a notice and comment rulemaking which specifies the following:
(i) Identification of the chemical for which testing is required under the rule.
(ii) The health or environmental effect or effects or other characteristics for which testing is being required.
(iii) Which test substance(s) must be tested.
(iv) Standards for the development of test data.
(v) The EPA Good Laboratory Practice requirements for the required testing.
(vi) Schedule for submission of interim reports and/or final reports to EPA.
(vii) Who must submit either letters of intent to conduct testing or exemption applications.
(viii) What types of data EPA will examine in determining equivalence if more than one test substance is to be tested.
(2) Under two-phase test rule development, EPA will promulgate a Phase I test rule in part 799 of this chapter through a notice and comment rulemaking which specifies the following:
(i) Identification of the chemical for which testing is required under the rule.
(ii) The health or environmental effect or effects or other characteristics for which testing is being required.
(iii) Which test substance(s) must be tested.
(iv) A reference to appropriate guidelines for the development of test data.
(v) The EPA Good Laboratory Practice requirements for the required testing.
(vi) Who must submit either letters of intent to conduct testing and study plans, or exemption applications.
(vii) What types of data EPA will examine in determining equivalence if more than one test substance is to be tested.
(3) Under two-phase test rule development, test standards and schedules will be developed in a second phase of rulemaking as described in §§ 790.50 and 790.52.
§ 790.42 Persons subject to a test rule.
(a) Each test rule described in § 790.40 will specify whether manufacturers, processors, or both are subject to the requirement for testing of the subject chemical under section 4(b)(3)(B) of the Act and will indicate who will be required to submit letters of intent to conduct testing.
(1) If testing is being required to allow evaluation of risks:
(i) Primarily associated with manufacture of the chemical, or
(ii) Associated with both manufacturer and processing of the chemical, or
(iii) Associated with distribution in commerce, use, and/or disposal activities concerning the chemical, each manufacturer of the chemical will be subject and must comply with the requirements of the test rule.
(2) While legally subject to the test rule in circumstances described in paragraphs (a)(1) (ii) and (iii) of this section, processors of the chemical must comply with the requirements of the test rule only if processors are directed to do so in a subsequent notice as set forth in § 790.48(b).
(3) If testing is being required to allow evaluation of risks associated solely with processing of the chemical, processors will be subject and must comply with the requirements of the test rule.
(4) While legally subject to the test rule in circumstances described in paragraph (a)(1) of this section, persons who manufacture less than 500 kg (1,100 lb) of the chemical annually during the period from the effective date of the test rule to the end of the reimbursement period, must comply with the requirements of the test rule only if such manufacturers are directed to do so in a subsequent notice as set forth in § 790.48, or if directed to do so in a particular test rule.
(5) While legally subject to the test rule in circumstances described in paragraph (a)(1) of this section, persons who manufacture small quantities of the chemical solely for research and development (meaning quantities that are not greater than those necessary for purposes of scientific experimentation or analysis or chemical research on, or analysis of, such chemical or another chemical, including such research or analysis for development of a product) from the effective date of the test rule to the end of the reimbursement period, must comply with the requirements of the test rule only if such manufacturers are directed to do so in subsequent notice set forth in § 790.48, or if directed to do so in a particular test rule.
(6) If testing is being required to allow evaluation of risks associated primarily with manufacture of a chemical for research and development (R & D) purposes, manufacturers of the chemical for R & D will be subject and must comply with the requirements of the test rule.
(b) [Reserved]
§ 790.45 Submission of letter of intent to conduct testing or exemption application.
(a) No later than 30 days after the effective date of a test rule described in § 790.40, each person subject to that test rule and required to comply with the requirements of that test rule as provided in § 790.42(a) must, for each test required, send his or her notice of intent to conduct testing, or submit to EPA an application for exemption from testing by the method specified in § 790.5(b).
(b) EPA will consider letters of intent to test as commitments to sponsor the tests for which they are submitted unless EPA agrees to the substitution of an exemption application in instances where more than one person indicates an intent to sponsor equivalent tests.
(c) Each letter of intent to conduct testing must include:
(1) Identification of test rule.
(2) Name, address, and telephone number of the firm(s) which will be sponsoring the tests.
(3) Name, address, and telephone number of the appropriate individual to contact for further information.
(4) For sponsors participating in a testing consortium – a list of all members of the consortium, the signature of an authorized representative of each member, and a designation of who is to serve as principal sponsor.
(5) A list of the testing requirements for which the sponsor(s) intends to conduct tests.
(6) If EPA is requiring testing of more than one representative substance – which test substance the sponsor(s) intends to use in each of the tests.
(7) A payment identity number on the front page of the letter, as required in § 700.45(g)(4) of this chapter.
(d)(1) Any person not manufacturing or processing the subject chemical as of the effective date of the test rule describing in § 790.40 or by 30 days after the effective date of the rule who, before the end of the reimbursement period, manufacturers or processes the test chemical and who is subject to and required to comply with the requirements of the test rule must submit the letter of intent to test or an exemption application required by paragraph (a) of this section by the date manufacture or processing begins, or
(2) When both manufacturers and processors are subject to the rule, any person not processing the subject chemical as of the effective date of the test rule described in § 790.40 or by 30 days after publication of the
(e) Manufacturers subject to a test rule described in § 790.40 who do not submit to EPA either a letter of their intent to conduct tests or a request for an exemption from testing for each test for which testing is required in the test rule will be considered in violation of that rule beginning on the 31st day after the effective date of the test rule described in § 790.40 or on the date manufacture begins as described in paragraph (d) of this section.
(f) Processors subject to a test rule described in § 790.40 and required to comply with the requirements of test rule pursuant to § 790.42(a)(2) or a
(g) Manufacturers and processors subject to a test rule described in § 790.40 and required to comply with the requirements of that test rule as provided in § 790.42(a) must remit the applicable fee specified in § 700.45(c) of this chapter.
§ 790.48 Procedure if no one submits a letter of intent to conduct testing.
(a) If only manufacturers are subject to the rule. (1) This paragraph applies if testing is being required solely to allow evaluation of risks associated with manufacturing and the test rule described in § 790.40 states that manufacturers only are responsible for testing.
(2) If no manufacturer subject to the test rule has notified EPA of its intent to conduct one or more of the required tests within 30 days after the effective date of the test rule described in § 790.40, EPA will notify all manufacturers, including those described in § 790.42(a)(4) and (a)(5), by certified mail or by publishing a notice of this fact in the
(3) If no manufacturer submits a letter of intent to conduct one or more of the required tests within 30 days after receipt of the certified letter or publication of the
(b) If manufacturers and processors are subject to the rule. (1) This paragraph applies if testing is being required to allow evaluation of risks associated with manufacturing and processing or with distribution in commerce, use, or disposal of the chemical and the test rule described in § 790.40 states that manufacturers and processors are responsible for testing.
(2) If no manufacturer subject to the rule has notified EPA of its intent to conduct testing for one or more of the required tests within 30 days after the effective date of the test rule described in § 790.40, EPA will publish a notice in the
(3) No later than 30 days after the date of publication of the
(4) If no manufacturer or processor of the test chemical has submitted a letter of intent to conduct one or more of the required tests within 30 days after the date of publication of the
(5) If no manufacturer or processor submits a letter of intent to EPA through CDX within 30 days after either receipt of the certified letter or publication in the
(c) Only processors are subject to the rule. (1) This paragraph applies if testing is being required solely to allow evaluation of risks associated with processing and the test rule described in § 790.40 states that only processors are responsible for testing.
(2) If no processor subject to the test rule has notified EPA through CDX of its intent to conduct one or more of the required tests within 30 days after the effective date of the test rule described in § 790.40, EPA will notify all the processors by certified mail or publish a notice in the
(3) If no processor submits a letter of intent through CDX to conduct one or more of the required tests within 30 days after receipt of the certified letter or publication of the
§ 790.50 Submission of study plans.
(a) Who must submit study plans. (1) Persons who notify EPA of their intent to conduct tests in compliance with the requirements of a single phase test rule as described in § 790.40(b)(1) must submit study plans for those tests prior to the initiation of each of these tests, unless directed by a particular test rule or consent agreement to submit study plans at a specific time.
(2) Persons who notify EPA of their intent to conduct tests in compliance with the requirements of a Phase I test rule as described in § 790.40(b)(2) must submit the proposed study plans for those tests on or before 90 days after the effective date of the Phase I rule; or, for processors complying with the notice described in § 790.48(b)(2), 90 days after the publication date of that notice; or 60 days after the date manufacture or processing begins as described in § 790.45(d), as appropriate, to the address in § 790.5(b).
(3) Study plans must be prepared according to the requirements of this subpart B and part 792 of this chapter. Only one set of study plans should be prepared and submitted by persons who are jointly sponsoring testing.
(4) Any person subject to a test rule may submit a study plan for any test required by the rule at any time, regardless of whether the person previously submitted an application for exemption from testing for that test.
(5) Unless EPA has granted an extension of time for submission of proposed study plans, manufacturers who notify EPA that they intend to conduct testing in compliance with the requirements of a Phase I test rule as described in § 790.40(b)(2) and who do not submit proposed study plans for those tests on or before 90 days after the effective date of the Phase I test rule or 60 days after the date manufacture begins as described in § 790.45(d) will be considered in violation of the test rule as if no letter of intent to test had been submitted.
(6) Unless EPA has granted an extension of time for submission of proposed study plans, processors who notify EPA that they intend to conduct testing in compliance with the requirements of a Phase I test rule as described in § 790.40(b)(2) and who do not submit proposed study plans for those tests on or before 90 days after the effective date of the Phase I test rule or 90 days after the publication date of the notice described in § 790.48(b)(2), or 60 days after the date processing begins as described in § 790.45(d), as appropriate, will be considered in violation of the test rule as if no letter of intent to test had been submitted.
(b) Extensions of time for submission of study plans. (1) EPA may grant requests for additional time for the development of study plans on a case-by-case basis. Requests for additional time for study plan development must be submitted to EPA by the method specified in § 790.5(b). Any extension request must state why EPA should grant the extension.
(2) Under two-phase rulemaking, extension requests must be submitted to EPA within 60 days after the effective date of the Phase I test rule as described in § 790.40(b)(2); or for processors complying with the notice described in § 790.48(b)(2), 60 days after the publication date of that notice; or 30 days after the date manufacture or processing begins as described in § 790.45(d), as appropriate.
(3) EPA will notify the submitter by certified mail of EPA’s decision to grant or deny an extension request.
(4) Persons who have been granted an extension of time for submission of study plans as described in paragraph (b)(1) of this section and who do not submit proposed study plans in compliance with the requirements of a Phase I test rule in accordance with the new deadline granted by EPA will be considered in violation of the test rule as if no letter of intent to test had been submitted as described in § 790.45(e) and (f).
(c) Content of study plans. (1) All study plans are required to contain the following information:
(i) Identity of the test rule.
(ii) The specific test requirements of that rule to be covered by the study plan.
(iii)(A) The names and addresses of the test sponsors.
(B) The names, addresses, and telephone numbers of the responsible administrative officials and project manager(s) in the principal sponsor’s organization.
(C) The name, address, and telephone number of the appropriate individual to contact for oral and written communications with EPA.
(D)(1) The names and addresses of the testing facilities and the names, addresses, and telephone numbers of the testing facilities’ administrative officials and project manager(s) responsible for the testing.
(2) Brief summaries of the training and experience of each professional involved in the study, including study director, veterinarian(s), toxicologist(s), pathologist(s), chemist(s), microbiologist(s), and laboratory assistants.
(iv) Identity and data on the chemical substance(s) being tested, including physical constants, spectral data, chemical analysis, and stability under test and storage conditions, as appropriate.
(v) Study protocol, including the rationale for any combination of test protocols; the rationale for species/strain selection; dose selection (and supporting data); route(s) or method(s) of exposure; description of diet to be used and its source; including nutrients and contaminants and their concentrations; for in vitro test systems, a description of culture medium and its source; and a summary of expected spontaneous chronic diseases (including tumors), genealogy, and life span.
(vi) Schedule for initiation and completion of each short-term test and of each major phase of long-term tests; dates for submission of interim progress and final reports to EPA that are within the reporting deadlines specified by EPA In the final test rule.
(2) Information required in paragraph (c)(1)(iii)(D) of this section is not required in proposed study plans submitted in compliance with the requirements of a Phase I test rule if the information is not available at the time of study plan submission; however, the information must be submitted before the initiation of testing.
(d) Incomplete study plans. (1) Upon receipt of a study plan, EPA will review the study plan to determine whether it complies with paragraph (c) of this section. If EPA determines that the study plan does not comply with paragraph (c) of this section, EPA will notify the submitter that the submission is incomplete and will identify the deficiencies and the steps necessary to complete the submission.
(2) The submitter will have 15 days after the day it receives this notice to submit appropriate information to make the study plan complete.
(3) If the submitter fails to provide appropriate information to complete a proposed study plan submitted in compliance with the requirements of a Phase I test rule on or before 15 days after receipt of the notice, the submitter will be considered in violation of the test rule as if no letter of intent to conduct the test had been submitted as described in § 790.45(e) and (f).
(e) Amendments to study plans. Test sponsors must submit all amendments by the method specified in § 790.5(b).
§ 790.52 Phase II test rule.
(a) If EPA determines that the proposed study plan described in § 790.50(a)(2) complies with § 790.50(c), EPA will publish a proposed Phase II test rule in the
(b) EPA will provide a 45-day comment period and will provide an opportunity for an oral presentation upon the request of any person. EPA may extend the comment period if it appears from the nature of the issues raised by EPA’s review or from public comments that further comment is warranted.
(c) After receiving and considering public comments on the study plan, EPA will adopt, as proposed or as modified in response to EPA review and public comments, the study protocol section of the study plan, as defined by § 790.50(c)(1)(v) of this chapter, as the test standard for the required testing, and the schedule section of the study plan, as defined by § 790.50(c)(1)(vi) of this chapter, as the schedule for the required testing in a final Phase II test rule.
§ 790.55 Modification of test standards or schedules during conduct of test.
(a) Application. Any test sponsor who wishes to modify the test schedule for the mandatory testing conditions or requirements (i.e., “shall statements”) in the test standard for any test required by a test rule must submit an application in accordance with this paragraph. Application for modification must be made by the method specified in § 790.5(b). Applications must include an appropriate explanation and rationale for the modification. Where a test sponsor requests EPA to provide guidance or to clarify a non-mandatory testing requirement (i.e., “should statements”) in a test standard, the test sponsor must submit these requests to EPA by the method format specified in § 790.5(b).
(b) Adoption. (1) Where EPA concludes that the requested modification of a test standard or schedule for a test required under a test rule is appropriate, EPA will proceed in accordance with this paragraph (b).
(2) Where, in EPA’s judgment, the requested modification of the test standard or schedule would not alter the scope of the test or significantly change the schedule for completing the test, EPA will not ask for public comment before approving the modification. EPA will notify the test sponsor by letter of EPA’s approval. EPA will place copies of each application and EPA approval letter in the rulemaking record for the test rule in question. EPA will publish a notice annually in the
(3) Where, in EPA’s judgment, the requested modification of a test standard or schedule would significantly alter the scope of the test or significantly change the schedule for completing the test, EPA will publish a notice in the
(4) For purposes of this paragraph (b), a requested modification of a test standard or schedule for a test required under a test rule would alter the scope of the test or significantly change the schedule for completing the test if the modification would:
(i) Change the test species.
(ii) Change the route of administration of the test chemical.
(iii) Change the period of time during which the test species is exposed to the test chemical.
(iv) Except as provided in paragraph (b)(3) of this section, extend the final reporting deadline more than 12 months from the date specified in the final rule.
(c) Disapproval. Where EPA concludes that the requested modification of a test standard or schedule for a test required under a test rule is not appropriate, EPA will so notify the test sponsor in writing.
(d) Timing. (1) Test sponsors should submit all applications for test schedule modifications at least 60 days before the reporting deadline for the test in question.
(2) EPA will not normally approve any test schedule extensions submitted less than 30 days before the reporting deadline for the test in question.
(3) Except as provided in paragraph (b)(3) of this section, EPA may grant extensions for up to 1 year but will normally limit extensions to a period of time equal to the in-life portion of the test plus 60 days.
(4) EPA will normally approve only one deadline extension for each test.
(5) Test sponsors should submit requests for test standard modifications as soon as they determine that the test cannot be successfully completed according to the test standard specified in the rule.
§ 790.59 Failure to comply with a test rule.
(a) Persons who notified EPA of their intent to conduct a test required in a test rule in part 799 of this chapter and who fail to conduct the test in accordance with the test standards and schedules adopted in the test rule, or as modified in accordance with § 790.55, will be in violation of the rule.
(b) Any person who fails or refuses to comply with any aspect of this part or a test rule under part 799 of this chapter is in violation of section 15 of the Act. EPA will treat violations of the Good Laboratory Practice standards as indicated in § 792.17 of this chapter.
(c) Persons who fail to pay the requisite fee as specified in § 700.45(c) of this chapter will be in violation of the rule.
Subpart D – Implementation, Enforcement and Modification of Consent Agreements
§ 790.60 Contents of consent agreements.
(a) Standard provisions. All consent agreements will contain the following provisions:
(1) Identification of the chemical(s) to be tested.
(2) The health effects, environmental effects and/or other characteristics for which testing will be required.
(3) The names and addresses of each manufacturer and/or processor who will sign the agreement.
(4) The name and address of the manufacturer, processor or other entity who has agreed to act as the principal test sponsor.
(5) The technical or commercial grade, level of purity or other characteristics of the test substances(s) or mixture(s).
(6) Standards for the development of test data.
(7) A requirement that testing will be conducted in accordance with the EPA Good Laboratory Practice (GLP) regulations (40 CFR part 792).
(8) Schedules with reasonable deadlines for submitting interim progress and/or final reports to EPA.
(9) A requirement that the principal sponsor will submit a study plan to EPA in accordance with § 790.62.
(10) A statement that the results of testing conducted pursuant to the consent agreement will be announced to the public in accordance with the procedures specified in section 4(d) of the Act and that the disclosure of data generated by such testing will be governed by section 14(b) of the Act.
(11) A requirement that the manufacturers and/or processors signing the consent agreement will comply with the notification requirements of section 12(b)(1) of the Act and part 707 of this chapter if they export or intend to export the substance or mixture for which the submission of data is required under the agreement and a statement that any other person who exports or intends to export such substance or mixture is subject to the above cited export notification requirements.
(12) A requirement that, in the event EPA promulgates a significant new use rule applicable to the test chemical under section 5(a)(2), the consent agreement will have the status of a test rule for purposes of section 5(b)(1)(A) and manufacturers and/or processors signing the agreement will comply with the data submission requirements imposed by that provision.
(13) A statement that each manufacturer and/or processor signing the agreement agrees that violation of its requirements will constitute a “prohibited act” under section 15(1) of the Act and will trigger all provisions of TSCA applicable to a violation of section 15.
(14) A statement that, in the event one or more provisions of the agreement are determined to be unenforceable by a court, the remainder of the agreement would not be presumed to be valid and EPA will then either initiate a rulemaking proceeding or publish in the
(15) A statement that the Agency may conduct laboratory inspections and/or study audits of the testing being conducted pursuant to the consent agreement in accordance with the authority and procedures contained in section 11 of the Act.
(16) A statement that EPA acceptance of a consent agreement constitutes “final agency action” for purposes of 5 U.S.C. 704.
(17) Any other requirements that the parties agree are necessary to achieve the purposes of the Act.
(18) Payment identity number, as required in § 700.45(g)(4) of this chapter.
(b) Contents of standards for the development of data. The standards for the development of the data included in consent agreements will be based on the TSCA test guidelines in 40 CFR parts 796, 797, and 798, the Organization for Economic Cooperation and Development (OECD) test guidelines, the EPA pesticide assessment guidelines published by The National Technical Information Service (NTIS), or other suitable test methodologies. During the negotiation of consent agreements, EPA will initially propose suitable test guidelines as the required test standards; manufacturers and processors or other interested parties may then suggest alternative methodologies or modifications to the Agency’s proposed guidelines. These alternative methodologies or modifications will be adopted only where, in the judgment of EPA, they will develop at least equally reliable and adequate data on the chemical substance or mixture subject to the agreement.
(c) Statement of rationale for consent agreement. EPA will prepare a written explanation of the basis for each consent agreement. This document will summarize the agreement, describe any ITC testing recommendations for the chemical involved, outline the chemical’s use and exposure characteristics, and explain the objectives of the testing to be conducted and the rationale for the specific studies selected. This document will be published in the
(d) Fees. Manufacturers and/or processors signing the consent agreement are subject to the applicable fee specified in § 700.45(c) of this chapter.
§ 790.62 Submission of study plans and conduct of testing.
(a) Timing of submission. The principal sponsor of testing conducted pursuant to a consent agreement shall submit a study plan no later than 45 days prior to the initiation of testing.
(b) Content of study plans. All study plans are required to contain the following information:
(1) Identity of the consent agreement under which testing will be performed.
(2) The specific test requirements to be covered by the study plan.
(3) The name and address of the principal test sponsor.
(4) The names, addresses, and telephone numbers of the responsible administrative official[s] and project manager[s] in the principal sponsor’s organization.
(5) The names, addresses, and telephone numbers of the technical contacts at each manufacturer and/or processor subject to the agreement.
(6) The names and addresses of the testing facilities responsible for the testing and the names, addresses, and telephone numbers of the administrative officials[s] and project manager[s] assigned to oversee the testing program at these facilities.
(7) Brief summaries of the training and experience of each professional involved in the study, including study director, veterinarian[s], toxicologist[s], pathologist[s], chemist[s], microbiologist[s], and laboratory assistants.
(8) Identity and supporting data on the chemical substance[s] being tested, including physical constants, spectral data, chemical analysis, and stability under test and storage conditions, as appropriate.
(9) Study protocol, including the rationale for any combination of test protocols; the rationale for species/strain selection; dose selection (and supporting data); route(s) or method(s) of exposure; description of diet to be used and its source, including nutrients and contaminants and their concentrations; for in vitro test systems, a description of culture medium and its source; and a summary of expected spontaneous chronic diseases (including tumors), genealogy, and life span.
(10) A schedule, with reasonable timeables and deadlines, for initiation and completion of each short-term test and of each major phases of long-term tests, and submission of interim progress and/or final reports to EPA.
(c) Review and modification. (1) Upon receipt of a study plan, EPA will review it to determine whether it complies with paragraph (b) of this section. If EPA determines that the study plan does not comply with paragraph (b) of this section, EPA will notify the submitter that the plan is incomplete and will identify the deficiencies and the steps necessary to complete the plan. It is the responsibility of the test sponsor to review the study protocols to determine if they comply with all the mandatory testing conditions and requirements in the test standards (i.e., “shall statements”).
(2) The submitter will have 15 days after the day it receives a notice under paragraph (c)(1) of this section to submit appropriate information to make the study plan complete.
(3) If the submitter fails to provide appropriate information to complete a study plan within 15 days after having received a notice under paragraph (c)(1) of this section, the submitter will be considered to be in violation of the consent agreement and subject to enforcement proceedings pursuant to § 790.65 (c) and (d).
(4) The test sponsor shall submit any amendments to study plans to EPA using the method specified in § 790.5(b).
(d) Functions of the principal test sponsor. When testing is being conducted pursuant to a consent agreement, the principal test sponsor will be responsible for submitting interim progress and final reports to EPA, informing the Agency of any proposed changes in standards for the development of data, study plans or testing schedules, and communicating with the Agency about laboratory inspections and other matters affecting the progress of testing.
§ 790.65 Failure to comply with a consent agreement.
(a) Manufacturers and/or processors who have signed a consent agreement and who fail to comply with the test requirements, test standards, GLP regulations, schedules, or other provisions contained in the consent agreement, or in modifications to the agreement adopted pursuant to § 790.68, will be in violation of the consent agreement.
(b) The Agency considers failure to comply with any aspect of a consent agreement, including the failure to pay requisite fees as specified in § 700.45 of this chapter, to be a “prohibited act” under section 15 of TSCA, subject to all the provisions of the Act applicable to violations of section 15. Section 15(1) of TSCA makes it unlawful for any person to fail or refuse to comply with any rule or order issued under section 4. Consent agreements adopted pursuant to this part are “orders issued under section 4” for purposes of section 15(1) of TSCA.
(c) Manufacturers and/or processors who violate consent agreements are subject to criminal and/or civil liability. Under the penalty provisions of section 16 of TSCA, such firms could be subject to a civil penalty of up to $25,000 per violation with each day in violation constituting a separate violation of section 15. Intentional violations could lead to the imposition of criminal penalties of up to $25,000 for each day of violation and imprisonment for up to one year. In addition, EPA could invoke the remedies available under section 17 of TSCA, including seeking an injunction to compel adherence to the requirements of the consent agreement.
(d) Noncompliance with a consent agreement will constitute conduct “in violation of this Act” under section 20(a)(1) of TSCA. Thus, failure to comply with the requirements of a consent agreement could result in a citizens’ civil action under section 20(a)(1) of TSCA.
§ 790.68 Modification of consent agreements.
(a) Changes in the scope of testing. (1) Manufacturers or processors subject to a consent agreement, other persons or EPA may seek modifications in the scope of testing performed under the consent agreement. If, upon receiving a request for modification, EPA determines that new issues have been raised that warrant reconsideration of the scope of testing, or if EPA determines on its own that such reconsideration is appropriate, EPA will publish a
(2) The scope of testing required by a consent agreement will be modified only where there is a consensus concerning the modified testing requirements among EPA, affected manufacturers and/or processors, and other persons who have asked to participate in or monitor negotiations under paragraph (a)(1) of this section. In determining whether a consensus exists, EPA will employ the criteria specified in § 790.22(b)(8). In the absence of consensus, EPA may initiate rulemaking under section 4(a) of the Act if it concludes that any testing beyond that required by the consent agreement is necessary and that the other statutory findings required by section 4(a) can be made. While such rulemaking proceedings are underway, the consent agreement will remain in effect unless EPA finds that the testing required by the agreement is or may be unnecessary in view of the testing requirements included in EPA’s proposed rule.
(b) Changes in test standards or schedules. (1) Any test sponsor who wishes to modify the test schedule for any test required under a consent agreement must submit an application in accordance with this paragraph. Application for modification must be made using the method specified in § 790.5(b). Applications must include an appropriate explanation and rationale for the modification. EPA will consider only those applications that request modifications to mandatory testing conditions or requirements (“shall statements” in the consent agreement). Where a test sponsor requests EPA to provide guidance or to clarify a non-mandatory testing requirement (i.e., “should statements”), the test sponsor shall submit these requests to EPA using the method specified in § 790.5(b).
(2)(i) Where EPA concludes that the requested modification of a test standard or schedule for a test required under a consent agreement is appropriate, EPA will proceed in accordance with this paragraph (b)(2).
(ii) Where, in EPA’s judgment, the requested modification of a test standard or schedule would not alter the scope of the test or significantly change the schedule for completing the test, EPA will not ask for public comment before approving the modification. EPA will notify the test sponsor, and any other persons who have signed the consent agreement, by letter of EPA’s approval. EPA will place copies of each application and EPA approval letter in the administrative record maintained for the consent agreement in question. EPA will publish a notice annually in the
(iii) Where, in EPA’s judgment, the requested modification of a test standard or schedule would significantly alter the scope of the test or significantly change the schedule for completing the test, EPA will publish a notice in the
(iv) For purposes of this paragraph (b)(2), a requested modification of a test standard of schedule for a test required under a consent agreement would alter the scope of the test or significantly change the schedule for completing the test if the modification would:
(A) Change the test species.
(B) Change the route of administration of the test chemical.
(C) Change the period of time during which the test species is exposed to the test chemical.
(D) Except as provided in paragraph (b)(2)(iii) of this section, extend the final reporting deadline more than 12 months from the date specified in the consent order.
(3) Where EPA concludes that the requested modification of a test standard or schedule for a test requirement under a consent agreement is not appropriate, EPA will so notify the test sponsor in writing.
(c) Timing. (1) Test sponsors should submit all applications for test schedule modifications at least 60 days before the reporting deadline for the test in question.
(2) EPA will not normally approve any test schedule extensions submitted less than 30 days before the reporting deadline for the test in question.
(3) Except as provided in paragraph (b)(2)(iii) of this section, EPA may grant extensions as shown necessary for up to 1 year but will normally limit extensions to a period of time equal to the in-life portion of the test plus 60 days.
(4) EPA will normally approve only one deadline extension for each test.
(5) Test sponsors should submit requests for test standard modifications as soon as they determine that the test cannot be successfully completed according to the test standard specified in the consent order.
Subpart E – Exemptions From Test Rules
§ 790.80 Submission of exemption applications.
(a) Who should file applications. (1) Any manufacturer or processor subject to a test rule in part 799 of this chapter may submit an application to EPA for an exemption from performing any or all of the tests required under the test rule.
(2) Processors will not be required to apply for an exemption or conduct testing unless EPA so specifies in a test rule or in a special
(i) If testing is being required to allow evaluation of risks associated with manufacturing and processing or with distribution in commerce, use, or disposal of the chemical and manufacturers do not submit notice(s) of intent to conduct the required testing; or
(ii) If testing is being required solely to allow evaluation of risks associated with processing of the chemical.
(b) When applications must be filed. (1) Exemption applications must be filed within 30 days after the effective date of the test rule described in § 790.40 or, if being submitted in compliance with the
(2) Exemption applications must be filed by the date manufacture or processing begins by any person not manufacturing or processing the subject chemical as of the effective date of the test rule described in § 790.40 or by 30 days after the effective date of the test rule described in § 790.40, who, before the end of the reimbursement period, manufactures or processes the test substance and who is subject to the requirement to submit either a letter of intent to test or an exemption application.
(3) When both manufacturers and processors are subject to the rule, exemption applications must be filed by the date processing begins by any person not processing as of the effective date of the test rule described in § 790.40 or by 30 days after publication of the
(c) Scope of application. A person may apply for an exemption from all, or one or more, specific testing requirements in a test rule in part 799 of this chapter.
§ 790.82 Content of exemption application.
The exemption application must contain:
(a) The identity of the test rule, the chemical identity, and the CAS No. of the test substance on which the application is based.
(b) The specific testing requirement(s) from which an exemption is sought and the basis for the exemption request.
(c) Name, address, and telephone number of applicant.
(d) Name, address, and telephone number of appropriate individual to contact for further information.
(e)(1) If required in the test rule to establish equivalence:
(i) The chemical identity of the test substance on which the application is based.
(ii) Equivalence data specified in § 790.85.
(2) If a test rule requires testing of a single representative substance, EPA will consider all forms of the chemical subject to that rule to be equivalent and will not require the submission of equivalence data as described in § 790.85.
§ 790.85 Submission of equivalence data.
If EPA requires in a test rule promulgated under section 4 of the Act the testing of two or more test substances which are forms of the same chemical, each exemption applicant must submit the following data:
(a) The chemical identity of each technical-grade chemical substance or mixture manufactured and/or processed by the applicant for which the exemption is sought. The exact type of identifying data required will be specified in the test rule, but may include all characteristics and properties of the applicant’s substance or mixture, such as boiling point, melting point, chemical analysis (including identification and amount of impurities), additives, spectral data, and other physical or chemical information that may be relevant in determining whether the applicant’s substance or mixture is equivalent to the specific test substance.
(b) The basis for the applicant’s belief that the substance or mixture is equivalent to the test substance or mixture.
(c) Any other data which exemption applicants are directed to submit in the test rule which may bear on a determination of equivalence. This may include a description of the process by which each technical-grade chemical substance or mixture for which an exemption is sought is manufactured or processed prior to use or distribution in commerce by the applicant.
§ 790.87 Approval of exemption applications.
(a) EPA will conditionally approve exemption applications if:
(1)(i) For single-phase test rules, EPA has received a letter of intent to conduct the testing from which exemption is sought;
(ii) For two-phase test rules, EPA has received a complete proposed study plan for the testing from which exemption is sought and has adopted the study plan, as proposed or modified, as test standards and schedules in a final Phase II test rule; and
(2) The chemical substance or mixture with respect to which the application was submitted is equivalent to a test substance or mixture for which the required data have been or are being submitted in accordance with a test rule; and
(3) Submission of the required test data concerning that chemical substance or mixture would be duplicative of data which have been or are being submitted to EPA in accordance with a test rule.
(b)(1) If a single representative substance is to be tested under a test rule, EPA will consider all forms of the chemical subject to that rule to be equivalent and will contact the exemption applicant only if information is missing or unclear.
(2) If two or more representative substances are to be tested under a test rule, EPA will evaluate equivalence claims made in each exemption application according to the criteria discussed in the test rule.
(i) If EPA finds an equivalence claim to be in error or inadequately supported, the applicant will be notified by certified mail. The applicant will be given 15 days to provide clarifying information.
(ii) Exemption applicants will be notified that equivalence has been accepted or rejected.
(c)(1) EPA will give exemption applicants final notice that they have received a conditional exemption through one of the following ways:
(i) A final Phase II test rule that adopts the study plans in a two-phase rulemaking.
(ii) A separate
(iii) A letter by certified mail will give exemption applicants final notice that they have received a conditional exemption.
(2) All conditional exemptions thus granted are contingent upon the test sponsors’ successful completion of testing according to the specifications of the test rule.
§ 790.88 Denial of exemption application.
(a) EPA may deny any exemption application if:
(1) EPA determines that the applicant has failed to demonstrate that the applicant’s chemical is equivalent to the test substance; or
(2) The exemption applicant fails to submit any of the information specified in § 790.82; or
(3) The exemption applicant fails to submit any of the information specified in § 790.85 if required in the test rule; or
(4)(i) For single-phase test rules, EPA has not received a letter of intent to conduct the test for which exemption is sought; or
(ii) For two-phase test rules, EPA has not received an adequate study plan for the test for which exemption is sought; or
(5) The study sponsor(s) fails to initiate the required testing by the deadlines adopted in the test rule; or
(6) The study sponsor(s) fails to submit data as required in the test standard and deadlines for submission of test data as adopted in the test rule or as modified in accordance with § 790.55.
(b) EPA will notify the exemption applicant by certified mail or
§ 790.90 Appeal of denial of exemption application.
(a) Within 30 days after receipt of notification that EPA has denied an application for exemption, the applicant may file an appeal with EPA.
(b) The appeal shall indicate the basis for the applicant’s request for reconsideration.
(c)(1) The applicant may also include a request for a hearing. Hearings will be held according to the procedures described in § 790.97.
(2) Hearing requests must be submitted using the method specified in § 790.5(b) and be received by EPA within 30 days of receipt of the Agency’s notification under § 790.88(b). Hearing requests must provide reasons why a hearing is necessary.
(d) If EPA determines that there are material issues of fact, then the request for a hearing will be granted. If EPA denies a hearing request, EPA will base its decision on the written submission.
(e) EPA will notify the applicant of its decision within 60 days after EPA receives the appeal described in paragraph (a) of this section or within 60 days after completion of a hearing described in paragraph (c) of this section.
(f) The filing of an appeal from the denial of an exemption shall not act to stay the applicant’s legal obligations under a test rule promulgated under section 4 of the Act.
§ 790.93 Termination of conditional exemption.
(a) EPA shall terminate a conditional exemption if it determines that:
(1) The test which provided the basis for approval of the exemption application has not been started by the deadlines for initiation of testing adopted in the test rule or modified in accordance with § 790.55; or
(2) Data required by the test rule have not been generated in accordance with the test standards or submitted in accordance with the deadlines for submission of test data that were adopted in the test rule or modified in accordance with § 790.55; or
(3) The testing has not been conducted or the data have not been generated in accordance with the Good Laboratory Practice requirements in part 792 of this chapter.
(b) If EPA determines that one or more of the criteria listed in paragraph (a) of this section has been met, EPA will notify each holder of an affected conditional exemption by certified mail or
(c) Within 30 days after receipt of a letter notification or publication of a notice in the
(d)(1) The exemption holder may also include a request for a hearing. Hearings will be held in accordance with the procedures set forth in § 790.97.
(2) Hearing requests must be submitted using the method specified in § 790.5(b) and must be received by EPA within 30 days after receipt of the letter or publication in the
(e) EPA will notify the exemption holder by certified letter or by
§ 790.97 Hearing procedures.
(a) Hearing requests must be submitted using the method specified in § 790.5(b). Such requests must include the applicant’s basis for appealing EPA’s decision.
(b) If more than one applicant has requested a hearing on similar grounds, all of those appeals will be considered at the same hearing unless confidentiality claims preclude a joint hearing.
(c) EPA will notify each applicant of EPA’s decision within 60 days after the hearing.
§ 790.99 Statement of financial responsibility.
Each applicant for an exemption shall submit the following sworn statement with his or her application:
I understand that if this application is granted before the reimbursement period described in section 4(c)(3)(B) of TSCA expires, I must pay fair and equitable reimbursement to the person or persons who incurred or shared in the costs of complying with the requirement to submit data and upon whose data the granting of my application was based.
PART 791 – DATA REIMBURSEMENT
Subpart A – General Provisions
(a) This part establishes procedures and criteria to be used in determining fair amounts of reimbursement for testing costs incurred under section 4(a) of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2603(a)).
(b) Section 4(c) of TSCA requires EPA to develop rules for the determination of fair and equitable reimbursement (15 U.S.C. 2603 (c)).
§ 791.2 Applicability.
(a) This rule is potentially applicable to all manufacturers, importers and processors who may be required by a specific test rule promulgated under section 4(a) of TSCA to conduct tests and submit data, and who seek the assistance of the Administrator in determining the amount or method of reimbursement. Persons subject to a test rule have an obligation from the date the test rule becomes effective until the end of the reimbursement period, either to test or to obtain an exemption and pay reimbursement.
(b) The provisions of this rule will take effect only when private efforts to resolve a dispute have failed and a manufacturer or processor requests EPA’s assistance.
§ 791.3 Definitions.
Terms defined in the Act, and not explicitly defined herein, are used with the meanings given in the Act.
(a) The Act refers to the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601 et seq.).
(b) The Agency or EPA refers to the Environmental Protection Agency.
(c) Byproduct refers to a chemical substance produced without a separate commercial intent during the manufacture, processing, use or disposal of another chemical substance or mixture.
(d) Dispute refers to a present controversy between parties subject to a test rule over the amount or method of reimbursement for the cost of developing health and environmental data on the test chemical.
(e) Exemption holder refers to a manufacturer or processor, subject to a test rule, that has received an exemption under sections 4(c)(1) or 4(c)(2) of TSCA from the requirement to conduct a test and submit data.
(f) Impurity refers to a chemical substance unintentionally present with another chemical substance or mixture.
(g) A party refers to a person subject to a section 4 test rule, who:
(1) Seeks reimbursement from another person under these rules, or
(2) From whom reimbursement is sought under these rules.
(h) Reimbursement period refers to a period that begins when the data from the last non-duplicative test to be completed under a test rule is submitted to EPA and ends after an amount of time equal to that which had been required to develop that data or after 5 years, whichever is later.
(i) Small business refers to a manufacturer or importer whose annual sales, when combined with those of its parent company (if any) are less than $30 million.
(j) Test rule refers to a regulation ordering the development of data on health or environmental effects or chemical fate for a chemical substance or mixture pursuant to TSCA section 4(a).
Subpart B – Hearing Procedures
§ 791.20 Initiation of reimbursement proceeding.
(a) When persons subject to a test rule are unable to reach an agreement on the amount or method of reimbursement for test data development as described in TSCA section 4(c)(3)(A), any of them may initiate a proceeding by filing two signed copies of a request for a hearing with a regional office of the American Arbitration Association and mailing a copy of the request to EPA, and to each person from whom they seek reimbursement, or who seeks reimbursement from them.
(b) The request for hearing must contain the following:
(1) The names and addresses of the filing party and its counsel, if any.
(2) Identification of the test rule under which the dispute arose.
(3) A list of the parties from whom reimbursement is sought or who are seeking reimbursement, a brief description of the attempts to reach agreement and a concise explanation of the issues on which the parties are unable to agree.
(c) The request for a hearing shall be accompanied by the appropriate administrative fee, as provided in a current Fee Schedule of the American Arbitration Association.
§ 791.22 Consolidation of hearings.
(a) Promptly upon receipt of the request for a hearing, the Administrator will publish a notice in the
(b) Any other person wishing to participate in the hearing shall so notify EPA within 45 days of the
§ 791.27 Pre-hearing preparation.
(a) Responses to requests for hearings. After filing of the request for hearing, if any other party desires to file an answer it shall be made in writing and filed with the American Arbitration Association, and a copy thereof shall be mailed to the other parties within a period of fourteen days from the date of receiving the complete list of parties. After the hearing officer is appointed, however, no new or different claim may be submitted except with the hearing officer’s consent.
(b) Pre-hearing conference. At the request of the parties or at the discretion of the American Arbitration Association, a pre-hearing conference with a representative of the American Arbitration Association and the parties or their counsel will be scheduled in appropriate cases to arrange for an exchange of information and the stipulation of uncontested facts so as to expedite the proceedings.
(c) Fixing of locale. The parties may mutually agree on the locale where the hearing is to be held. If the locale is not designated within 45 days from the time the complete list of parties is received, the American Arbitration Association shall have power to determine the locale. Its decision shall be final and binding. If any party requests, and informs the other parties of its request, that the hearing be held in a specific locale and the other parties file no objection thereto within 14 days of the request, the locale shall be the one requested.
(d) Time and place. The hearing officer shall fix the time and place for each hearing. The American Arbitration Association will mail notice to each party at least 14 days in advance.
§ 791.29 Appointment of hearing officer.
(a) Qualifications of hearing officer. All hearing officers shall be neutral, subject to disqualification for the reasons specified in paragraph (f) of this section.
(b) Appointment from panel. Promptly after receiving the complete list of parties at the close of the notice period described in § 791.22, the American Arbitration Association shall submit simultaneously to each party to the dispute an identical list of names. Each party to the dispute shall have thirty days from the mailing date in which to cross off any names objected to, number the remaining names to indicate the order of preference, and return the list to the American Arbitration Association. If a party does not return the list within the time specified, all persons named therein shall be deemed acceptable to that party. From among the persons who have been approved on all lists, and in accordance with the designated order of mutual preference, the American Arbitration Association shall invite the acceptance of a hearing officer to serve. If the parties fail to agree upon any of the persons named, or if acceptable hearing officers are unable to act, or if for any other reason the appointment cannot be made from the submitted lists, the American Arbitration Association shall have the power to make the appointment without the submission of any additional list.
(c) Nationality of hearing officer in international dispute. If one of the parties is a national or resident of a country other than the United States, the hearing officer shall upon the request of any party, be appointed from among the nationals of a country other than that of the parties.
(d) Number of hearing officers. The dispute shall be heard and determined by one hearing officer unless the American Arbitration Association, in its discretion, directs that a greater number of hearing officers be appointed.
(e) Notice of appointment. Notice of the appointment of the hearing officer, together with a copy of these rules, and the signed acceptance of the hearing officer shall be filed prior to the opening of the first hearing.
(f) Disclosure and challenge procedure. A person appointed as hearing officer shall disclose to the American Arbitration Association any circumstances likely to affect impartiality, including any bias or any financial or personal interest in the result of the hearing or any past or present relationship with the parties or their counsel. Upon receipt of such information from such hearing officer or other source, the American Arbitration Association shall communicate such information to the parties, and, if it deems it appropriate to do so, to the hearing officer and others. Thereafter, the American Arbitration Association shall determine whether the hearing officer should be disqualified and shall inform the parties of its decision, which shall be conclusive.
(g) Vacancies. If any hearing officer should resign, die, withdraw, refuse, be disqualified or be unable to perform the duties of the office, the American Arbitration Association may, on proof satisfactory to it, declare the office vacant. Vacancies shall be filled in accordance with the applicable provisions of these rules and the matter shall be reheard unless the parties shall agree otherwise.
§ 791.30 Hearing procedures.
(a) Representation by counsel. Any party may be represented by counsel. A party intending to be so represented shall notify the other parties and the American Arbitration Association of the name and address of counsel at least 5 days prior to the date set for the hearing at which counsel is first to appear. When a hearing is initiated by counsel, or where an attorney replies for the other party, such notice is deemed to have been given.
(b) Stenographic record. The American Arbitration Association shall make the necessary arrangements for the taking of a stenographic record. The parties shall share the cost of such record.
(c) Attendance at hearings. The hearing officer shall have the power to require the exclusion of anyone, including a party or other essential person, during the testimony of any witness to protect confidential business information. It shall be discretionary with the hearing officer to determine the propriety of the attendance of any other person.
(d) Oaths. Hearing officers shall swear or affirm their neutrality and their dedication to a fair and equitable resolution. Witnesses shall swear or affirm that they are telling the truth.
(e) Order of proceedings. (1) A hearing shall be opened by the filing of the oath of the hearing officer and by the recording of the place, time and date of the hearing, the presence of the hearing officer and parties, and counsel, if any, and by the receipt by the hearing officer of the request for hearing and answer, if any.
(2) The hearing officer may, at the beginning of the hearing, ask for statements clarifying the issues involved.
(3) The party or parties seeking reimbursement shall then present a claim and proofs and witnesses, who shall submit to questions or other examination. The party or parties from whom reimbursement is sought shall then present a defense and proofs and witnesses, who shall submit to questions or other examination. The hearing officer has discretion to vary this procedure but shall afford full and equal opportunity to all parties for the presentation of any material or relevant proofs.
(4) Exhibits, when offered by any party, shall be received in evidence by the hearing officer. The names and addresses of all witnesses and exhibits in order received shall be made a part of the record.
(f) Hearing in the absence of a party. A hearing may proceed in the absence of any party which, after due notice, fails to be present or fails to obtain an adjournment. An award shall not be made solely on the default of a party. The hearing officer shall require the parties who are present to submit such evidence as the hearing officer may require for the making of an award.
(g) Evidence. (1) The parties may offer such evidence as they desire and shall produce such additional evidence as the hearing officer may deem necessary to an understanding and determination of the dispute. The hearing officer shall be the judge of the relevancy and materiality of the evidence offered and conformity to legal rules of evidence shall not be necessary. All evidence shall be taken in the presence of all the hearing officers and of all the parties, except where any of the parties is absent in default, has waived the right to be present, or has been excluded by the hearing officer to protect confidential business information.
(2) All documents not filed with the hearing officer at the hearing, but arranged for by agreement of the parties, shall be filed with the American Arbitration Association for transmission to the hearing officer, according to the agreed-upon schedule. All parties shall be afforded opportunity to examine such documents.
(h) Evidence by affidavit and filing of documents. The hearing officer shall receive and consider the evidence of witnesses by affidavit, but shall give it only such weight as the hearing officer deems it entitled to after consideration of any objections made to its admission.
(i) Closing of hearings. The hearing officer shall specifically inquire of all parties whether they have any further proofs to offer or witnesses to be heard. Upon receiving negative replies, the hearing officer shall declare the hearings closed and record the time of closing of the hearing. If briefs are to be filed, the hearings shall be declared closed as of the final date set by the hearing officer for the receipt of briefs. If documents are to be filed as provided for in paragraph (g)(2) of this section and the date set for their receipt is later than that set for the receipt of briefs, the later date shall be the date of closing the hearings.
(j) Reopening of hearings. The hearings may be reopened on the hearing officer’s own motion, or upon application of a party at any time before the award is made. If the reopening of the hearings would prevent the making of the award within the specified time the matter may not be reopened, unless the parties agree upon the extension of the time limit.
(k) Waiver of oral hearings. The parties may provide, by written agreement, for the waiver of oral hearings. If the parties are unable to agree as to the procedure, the American Arbitration Association shall specify a fair and equitable procedure.
(l) Waiver of rules. Any party who proceeds with the hearing after knowledge that any provision or requirement of these rules has not been complied with and who fails to state objection thereto in writing, shall be deemed to have waived the right to object.
(m) Extensions of time. The parties may modify any period of time by mutual agreement. The American Arbitration Association for good cause may extend any period of time established by these rules, except the time for making the award. (§ 791.37(a)) The American Arbitration Association shall notify the parties of any such extension of time and its reason therefor.
(n) Communication with hearing officer. There shall be no direct communication between the parties and a hearing officer other than at oral hearings. Any other oral or written communications from the parties to the hearing officer shall be directed to the American Arbitration Association for transmittal to the hearing officer.
§ 791.31 Expedited procedures.
Unless the American Arbitration Association in its discretion determines otherwise, the Expedited Procedures described in this section shall be applied in any case where the total claim of any party does not exceed $5,000, exclusive of interest and hearing costs, and may be applied in other cases if the parties agree.
(a) Application of rules. The expedited hearings will be conducted according to the same procedures as the regular ones, except for those specifically changed by the expedited rules in this section, § 791.31.
(b) Notice by telephone. The parties shall accept all notices from the American Arbitration Association by telephone. Such notices by the American Arbitration Association shall subsequently be confirmed in writing to the parties. Notwithstanding the failure to confirm in writing any notice or objection hereunder, the proceeding shall nonetheless be valid if notice or obligation has, in fact, been given by telephone.
(c) Appointment and qualifications of hearing officers. The American Arbitration Association shall submit simultaneously to each party to the dispute an identical list of five persons from which one hearing officer shall be appointed. Each party shall have the right to strike two names from the list on a peremptory basis. The list is returnable to the American Arbitration Association within 10 days from the date of mailing. If for any reasons the appointment cannot be made from the list, the American Arbitration Association shall have the authority to make the appointment without the submission of additional lists. Such appointment shall be subject to disqualification for the reasons specified in § 791.29(f). The parties shall be given notice by telephone by the American Arbitration Association of the appointment of the hearing officer. The parties shall notify the American Arbitration Association, by telephone, within 7 days of any objections to the hearing officer(s) appointed. Any objection by a party to such hearing officer shall be confirmed in writing to the American Arbitration Association with a copy to the other parties.
(d) Time and place of hearing. The hearing officer shall fix the date, time and place of the hearing. The American Arbitration Association will notify the parties by telephone, 7 days in advance of the hearing date. Formal notice of hearing will be sent by the American Arbitration Association to the parties.
(e) The hearing. Generally, the hearing shall be completed within 1 day. The hearing officer, for good cause shown, may schedule an additional hearing to be held within 5 days.
(f) Time of award. Unless otherwise agreed to by the parties, the Award shall be rendered not later than 15 business days from the date of the closing of the hearing.
§ 791.34 Serving of notice.
(a) Each party shall be deemed to have consented that any papers, notices or process necessary or proper for the initiation or continuation of a hearing under these rules and for any appeal to EPA or any court action in connection therewith may be served upon such party by mail addressed to such party or its attorney at its last known address or by personal service, within or without the state wherein the arbitration is to be held (whether such party be within or without the United States of America), provided that reasonable opportunity to be heard with regard thereto has been granted such party.
(b) The American Arbitration Association shall, upon the written request of a party, furnish to such party, at its expense, certified facsimiles of any papers in the American Arbitration Association’s possession that may be required in appeal to EPA or judicial proceedings relating to the hearing.
§ 791.37 The award.
(a) Time of award. The award shall be made promptly by the hearing officer and, unless otherwise agreed by the parties, no later than 30 days from the date of closing the hearings, or if oral hearings have been waived, from the date of transmitting the final statements and proofs to the hearing officer.
(b) Form of award. The award shall be in writing and shall be signed either by the sole hearing officer or by at least a majority if there is more than one. It shall contain a concise statement of its basis and rationale, and a timetable for payment of any ordered reimbursement.
(c) Delivery of award to parties. Parties shall accept as legal delivery of the award the delivery of the award or a true copy thereof by certified mail to the party at its last known address or to its attorney, or by personal service.
§ 791.39 Fees and expenses.
(a) Administrative fees. (1) As a not-for-profit organization, the American Arbitration Association shall prescribe an Administrative Fee Schedule and a Refund Schedule to compensate it for the cost of providing administrative services. The schedule in effect at the time of filing or the time of refund shall be applicable.
(2) The administrative fees shall be advanced by the initiating party or parties, subject to final apportionment by the hearing officer in the award. The administrative fee is increased by 10 percent of the original for each additional party.
(b) Expenses. All expenses of the hearing, including the cost of recording (though not transcribing) the hearing and required traveling and other expenses of the hearing officer and of American Arbitration Association representatives, and the expenses of any witness or the cost of any proofs produced at the direct request of the hearing officer, shall be borne equally by the parties, unless they agree otherwise, or unless the hearing officer, in the award, assesses such expenses or any part thereof against any specified party or parties.
(c) Hearing officer’s fee. Hearing officers will normally serve without a fee. In prolonged or special cases the American Arbitration Association in consultation with the Administrator may determine that payment of a fee by the parties is appropriate and may establish a reasonable amount, taking into account the extent of service by the hearing officer and other relevant circumstances of the case. Any arrangements for compensation shall be made through the American Arbitration Association and not directly between the parties and the hearing officer.
Subpart C – Basis for Proposed Order
§ 791.40 Basis for the proposed order.
(a) The hearing officer shall propose a fair and equitable amount of reimbursement. The formula in paragraph (b) of this section shall be presumed to be fair and equitable as applied to all persons subject to a test rule. However, the hearing officer has the discretion to modify the formula, or to use some other basis for allocation if necessary. Additional factors that may be taken into account include, but are not limited to, relative amounts of exposure attributable to each person and the effect of the reimbursement share on competitive position.
(b) In general, each person’s share of the test cost shall be in proportion to its share of the total production volume of the test chemical:
(c) The burden of proposing modifications to the formula shall lie with the party requesting the modification.
§ 791.45 Processors.
(a) Generally, processors will be deemed to have fulfilled their testing and reimbursement responsibilities indirectly, through higher prices passed on by those directly responsible, the manufacturers. There are three circumstances in which processors will have a responsibility to provide reimbursement directly to those paying for the testing:
(1) When a test rule or subsequent
(2) When one or more manufacturers demonstrate to the hearing officer that it is necessary to include processors in order to provide fair and equitable reimbursement in a specific case.
(3) When one or more processors voluntarily agree to reimburse manufacturers for a portion of test costs. Only those processors who volunteer will incur the obligation.
(b) A hearing including processors shall be initiated in the same way as those including only manufacturers. Voluntary negotiations must be attempted in good faith first, and the request for a hearing must contain the names of the parties and a description of the unsuccessful negotiations.
(c) When processors as well as manufacturers are required to provide reimbursement, the hearing officer will decide for each case how the reimbursement should be allocated among the participating parties. When a test rule is applicable solely to processors, the hearing officer will apply the formula to the amount of the test chemical purchased or processed.
§ 791.48 Production volume.
(a) Production volume will be measured over a period that begins one calendar year before publication of the final test rule in the
(b) For the purpose of determining fair reimbursement shares, production volume shall include amounts of the test chemical imported in bulk form and mixtures, and the total domestic production of the chemical including that produced as a byproduct. Impurities will not be included unless the test rule specifically includes them.
(c) Amounts of the test chemical manufactured for export will not be included unless covered by a finding under TSCA section 12(a)(2).
(d) Chemicals excluded from the jurisdiction of TSCA by section 3(2)(B) need not be included in the computation of production volume. (Chemicals used as intermediates to produce pesticides are covered by TSCA.)
(e) The burden of establishing the fact that particular amounts of the test chemical are produced for exempt purposes lies with the party seeking to exclude those amounts from the calculation of his production volume.
§ 791.50 Costs.
(a) All costs reasonable and necessary to comply with the test rule, taking into account the practices of other laboratories in conducting similar tests, are eligible for reimbursement. Necessary costs include:
(1) Direct and indirect costs of planning, conducting, analyzing and submitting the test results to EPA.
(2) A reasonable profit, and a reasonable rate of interest and depreciation on the tester’s initial capital investment.
(3) The cost of repeating or repairing tests where failure was demonstrably due to some cause other than negligence of the tester.
(b) Costs attributable to tests beyond those specified by EPA shall not be eligible for reimbursement under this rule.
§ 791.52 Multiple tests.
When more than one of a particular kind of test required by the test rule is performed, the additional costs will be shared among all those holding exemptions. The costs of all the tests will be added together and each exemption holder shall be responsible for a share of the total which is equal to its share of the total production of the test chemical. The exemption holders shall divide their shares between test sponsors in proportion to the costs of their respective tests. Those sponsoring a particular test do not have to obtain exemptions for that test and therefore do not have reimbursement responsibilities for the same tests done by others.
Subpart D – Review
§ 791.60 Review.
(a) The hearing officer’s proposed order shall become the final Agency order 30 days after issuance unless within the 30-day period one of the parties requests Agency review or the Administrator of his own initiative decides to review the proposed order.
(b) The proposed order may be reviewed upon the record of the hearing and the petitions for review. If necessary, the Administrator may order the transcription of the stenographic record of the hearing, written briefs, oral arguments or any other reasonable aids to making an equitable decision.
(c) The final Agency order may be reviewed in federal court as provided by 26 U.S.C. 2603(c).
Subpart E – Final Order
§ 791.85 Availability of final Agency order.
The final Agency order shall be available to the public for inspection and copying pursuant to 5 U.S.C. 552(a)(2), subject to necessary confidentiality restrictions.
Subpart F – Prohibited Acts
§ 791.105 Prohibited acts.
Failure to provide information required by the Agency or to pay the amounts awarded under this rule within time alloted in the final order shall constitute a violation of 15 U.S.C. 2614(1) or 2614(3).
PART 792 – GOOD LABORATORY PRACTICE STANDARDS
Subpart A – General Provisions
§ 792.1 Scope.
(a) This part prescribes good laboratory practices for conducting studies relating to health effects, environmental effects, and chemical fate testing. This part is intended to ensure the quality and integrity of data submitted pursuant to testing consent agreements and test rules issued under section 4 of the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2006, 15 U.S.C. 2603 et seq.).
(b) This part applies to any study described by paragraph (a) of this section which any person conducts, initiates, or supports on or after September 18, 1989.
(c) It is EPA’s policy that all data developed under section 5 of TSCA be in accordance with provisions of this part. If data are not developed in accordance with the provisions of this part, EPA will consider such data insufficient to evaluate the health and environmental effects of the chemical substances unless the submitter provides additional information demonstrating that the data are reliable and adequate.
§ 792.3 Definitions.
As used in this part the following terms shall have the meanings specified:
Batch means a specific quantity or lot of a test, control, or reference substance that has been characterized according to § 792.105(a).
Carrier means any material, including but not limited to, feed, water, soil, and nutrient media, with which the test substance is combined for administration to a test system.
Control substance means any chemical substance or mixture, or any other material other than a test substance, feed, or water, that is administered to the test system in the course of a study for the purpose of establishing a basis for comparison with the test substance for chemical or biologicaI measurements.
EPA means the U.S. Environmental Protection Agency.
Experimental start date means the first date the test substance is applied to the test system.
Experimental termination date means the last date on which data are collected directly from the study.
FDA means the U.S. Food and Drug Administration.
Person includes an individual, partnership, corporation, association, scientific or academic establishment, government agency, or organizational unit thereof, and any other legal entity.
Quality assurance unit means any person or organizational element, except the study director, designated by testing facility management to perform the duties relating to quality assurance of the studies.
Raw data means any laboratory worksheets, records, memoranda, notes, or exact copies thereof, that are the result of original observations and activities of a study and are necessary for the reconstruction and evaluation of the report of that study. In the event that exact transcripts of raw data have been prepared (e.g., tapes which have been transcribed verbatim, dated, and verified accurate by signature), the exact copy or exact transcript may be substituted for the original source as raw data. “Raw data” may include photographs, microfilm or microfiche copies, computer printouts, magnetic media, including dictated observations, and recorded data from automated instruments.
Reference substance means any chemical substance or mixture, or analytical standard, or material other than a test substance, feed, or water, that is administered to or used in analyzing the test system in the course of a study for the purposes of establishing a basis for comparison with the test substance for known chemical or biological measurements.
Specimen means any material derived from a test system for examination or analysis.
Sponsor means:
(1) A person who initiates and supports, by provision of financial or other resources, a study;
(2) A person who submits a study to the EPA in response to a TSCA section 4(a) test rule and/or a person who submits a study under a TSCA section 4 testing consent agreement or a TSCA section 5 rule or order to the extent the agreement, rule or order references this part; or
(3) A testing facility, if it both initiates and actually conducts the study.
Study means any experiment at one or more test sites, in which a test substance is studied in a test system under laboratory conditions or in the environment to determine or help predict its effects, metabolism, environmental and chemical fate, persistence, or other characteristics in humans, other living organisms, or media. The term “study” does not include basic exploratory studies carried out to determine whether a test substance or a test method has any potential utility.
Study completion date means the date the final report is signed by the study director.
Study director means the individual responsible for the overall conduct of a study.
Study initiation date means the date the protocol is signed by the study director.
Test substance means a substance or mixture administered or added to a test system in a study, which substance or mixture is used to develop data to meet the requirements of a TSCA section 4(a) test rule and/or is developed under a TSCA section 4 testing consent agreement or section 5 rule or order to the extent the agreement, rule or order references this part.
Test system means any animal, plant, microorganism, chemical or physical matrix, including but not limited to, soil or water, or components thereof, to which the test, control, or reference substance is administered or added for study. “Test system” also includes appropriate groups or components of the system not treated with the test, control, or reference substance.
Testing facility means a person who actually conducts a study, i.e., actually uses the test substance in a test system. “Testing facility” encompasses only those operational units that are being or have been used to conduct studies.
TSCA means the Toxic Substances Control Act (15 U.S.C, 2601 et seq.)
Vehicle means any agent which facilitates the mixture, dispersion, or solubilization of a test substance with a carrier.
§ 792.10 Applicability to studies performed under grants and contracts.
When a sponsor or other person utilizes the services of a consulting laboratory, contractor, or grantee to perform all or a part of a study to which this part applies, it shall notify the consulting laboratory, contractor, or grantee that the service is, or is part of, a study that must be conducted in compliance with the provisions of this part.
§ 792.12 Statement of compliance or non-compliance.
Any person who submits to EPA a test required by a testing consent agreement or a test rule issued under section 4 of TSCA shall include in the submission a true and correct statement, signed by the sponsor and the study director, of one of the following types:
(a) A statement that the study was conducted in accordance with this part; or
(b) A statement describing in detail all differences between the practices used in the study and those required by this part; or
(c) A statement that the person was not a sponsor of the study, did not conduct the study, and does not know whether the study was conducted in accordance with this part.
§ 792.15 Inspection of a testing facility.
(a) A testing facility shall permit an authorized employee or duly designated representative of EPA or FDA, at reasonable times and in a reasonable manner, to inspect the facility and to inspect (and in the case of records also to copy) all records and specimens required to be maintained regarding studies to which this part applies. The records inspection and copying requirements shall not apply to quality assurance unit records of findings and problems, or to actions recommended and taken, except the EPA may seek production of these records in litigation or formal adjudicatory hearings.
(b) EPA will not consider reliable for purposes of showing that a chemical substance or mixture does not present a risk of injury to health or the environment any data developed by a testing facility or sponsor that refuses to permit inspection in accordance with this part. The determination that a study will not be considered reliable does not, however, relieve the sponsor of a required test of any obligation under any applicable statute or regulation to submit the results of the study to EPA.
(c) Since a testing facility is a place where chemicals are stored or held, it is subject to inspection under section 11 of TSCA.
§ 792.17 Effects of non-compliance.
(a) The sponsor or any other person who is conducting or has conducted a test to fulfill the requirements of a testing consent agreement or a test rule issued under section 4 of TSCA will be in violation of section 15 of TSCA if:
(1) The test is not being or was not conducted in accordance with any requirement of this part;
(2) Data or information submitted to EPA under this part (including the statement required by § 792.12) include information or data that are false or misleading, contain significant omissions, or otherwise do not fulfill the requirements of this part; or
(3) Entry in accordance with § 792.15 for the purpose of auditing test data or inspecting test facilities is denied. Persons who violate the provisions of this part may be subject to civil or criminal penalties under section 16 of TSCA, legal action in United States district court under section 17 of TSCA, or criminal prosecution under 18 U.S.C. 2 or 1001.
(b) EPA, at its discretion, may not consider reliable for purposes of showing that a chemical substance or mixture does not present a risk of injury to health or the environment any study which was not conducted in accordance with this part. EPA, at its discretion, may rely upon such studies for purposes of showing adverse effects. The determination that a study will not be considered reliable does not, however, relieve the sponsor of a required test of the obligation under any applicable statute or regulation to submit the results of the study to EPA.
(c) If data submitted to fulfill a requirement of a testing consent agreement or a test rule issued under section 4 of TSCA are not developed in accordance with this part, EPA may determine that the sponsor has not fulfilled its obligations under section 4 of TSCA and may require the sponsor to develop data in accordance with the requirements of this part in order to satisfy such obligations.
Subpart B – Organization and Personnel
§ 792.29 Personnel.
(a) Each individual engaged in the conduct of or responsible for the supervision of a study shall have education, training, and experience, or combination thereof, to enable that individual to perform the assigned functions.
(b) Each testing facility shall maintain a current summary of training and experience and job description for each individual engaged in or supervising the conduct of a study.
(c) There shall be a sufficient number of personnel for the timely and proper conduct of the study according to the protocol.
(d) Personnel shall take necessary personal sanitation and health precautions designed to avoid contamination of test, control, and reference substances and test systems.
(e) Personnel engaged in a study shall wear clothing appropriate for the duties they perform. Such clothing shall be changed as often as necessary to prevent microbiological, radiological, or chemical contamination of test systems and test, control, and reference substances.
(f) Any individual found at any time to have an illness that may adversely affect the quality and integrity of the study shall be excluded from direct contact with test systems, test, control, and reference substances and any other operation or function that may adversely affect the study until the condition is corrected. All personnel shall be instructed to report to their immediate supervisors any health or medical conditions that may reasonably be considered to have an adverse effect on a study.
§ 792.31 Testing facility management.
For each study, testing facility management shall:
(a) Designate a study director as described in § 792.33 before the study is initiated.
(b) Replace the study director promptly if it becomes necessary to do so during the conduct of a study.
(c) Assure that there is a quality assurance unit as described in § 792.35.
(d) Assure that test, control, and reference substances or mixtures have been appropriately tested for identity, strength, purity, stability, and uniformity, as applicable.
(e) Assure that personnel, resources, facilities, equipment, materials and methodologies are available as scheduled.
(f) Assure that personnel clearly understand the functions they are to perform.
(g) Assure that any deviations from these regulations reported by the quality assurance unit are communicated to the study director and corrective actions are taken and documented.
§ 792.33 Study director.
For each study, a scientist or other professional of appropriate education, training, and experience, or combination thereof, shall be identified as the study director. The study director has overall responsibility for the technical conduct of the study, as well as for the interpretation, analysis, documentation, and reporting of results, and represents the single point of study control. The study director shall assure that:
(a) The protocol, including any change, is approved as provided by § 792.120 and is followed.
(b) All experimental data, including observations of unanticipated responses of the test system are accurately recorded and verified.
(c) Unforeseen circumstances that may affect the quality and integrity of the study are noted when they occur, and corrective action is taken and documented.
(d) Test systems are as specified in the protocol.
(e) All applicable good laboratory practice regulations are followed.
(f) All raw data, documentation, protocols, specimens, and final reports are transferred to the archives during or at the close of the study.
§ 792.35 Quality assurance unit.
(a) A testing facility shall have a quality assurance unit which shall be responsible for monitoring each study to assure management that the facilities, equipment, personnel, methods, practices, records, and controls are in conformance with the regulations in this part. For any given study, the quality assurance unit shall be entirely separate from and independent of the personnel engaged in the direction and conduct of that study. The quality assurance unit shall conduct inspections and maintain records appropriate to the study.
(b) The quality assurance unit shall:
(1) Maintain a copy of a master schedule sheet of all studies conducted at the testing facility indexed by test substance and containing the test system, nature of study, date study was initiated, current status of each study, identity of the sponsor, and name of the study director.
(2) Maintain copies of all protocols pertaining to all studies for which the unit is responsible.
(3) Inspect each study at intervals adequate to ensure the integrity of the study and maintain written and properly signed records of each periodic inspection showing the date of the inspection, the study inspected, the phase or segment of the study inspected, the person performing the inspection, findings and problems, action recommended and taken to resolve existing problems, and any scheduled date for re-inspection. Any problems which are likely to affect study integrity found during the course of an inspection shall be brought to the attention of the study director and management immediately.
(4) Periodically submit to management and the study director written status reports on each study, noting any problems and the corrective actions taken.
(5) Determine that no deviations from approved protocols or standard operating procedures were made without proper authorization and documentation.
(6) Review the final study report to assure that such report accurately describes the methods and standard operating procedures, and that the reported results accurately reflect the raw data of the study.
(7) Prepare and sign a statement to be included with the final study report which shall specify the dates inspections were made and findings reported to management and to the study director.
(c) The responsibilities and procedures applicable to the quality assurance unit, the records maintained by the quality assurance unit, and the method of indexing such records shall be in writing and shall be maintained. These items including inspection dates, the study inspected, the phase or segment of the study inspected, and the name of the individual performing the inspection shall be made available for inspection to authorized employees or duly designated representatives of EPA or FDA.
(d) An authorized employee or a duly designated representative of EPA or FDA shall have access to the written procedures established for the inspection and may request testing facility management to certify that inspections are being implemented, performed, documented, and followed up in accordance with this paragraph.
Subpart C – Facilities
§ 792.41 General.
Each testing facility shall be of suitable size and construction to facilitate the proper conduct of studies. Testing facilities which are not located within an indoor controlled environment shall be of suitable location to facilitate the proper conduct of studies. Testing facilities shall be designed so that there is a degree of separation that will prevent any function or activity from having an adverse effect on the study.
§ 792.43 Test system care facilities.
(a) A testing facility shall have a sufficient number of animal rooms or other test system areas, as needed, to ensure: proper separation of species or test systems, isolation of individual projects, quarantine or isolation of animals or other test systems, and routine or specialized housing of animals or other test systems.
(1) In tests with plants or aquatic animals, proper separation of species can be accomplished within a room or area by housing them separately in different chambers or aquaria. Separation of species is unnecessary where the protocol specifies the simultaneous exposure of two or more species in the same chamber, aquarium, or housing unit.
(2) Aquatic toxicity tests for individual projects shall be isolated to the extent necessary to prevent cross-contamination of different chemicals used in different tests.
(b) A testing facility shall have a number of animal rooms or other test system areas separate from those described in paragraph (a) of this section to ensure isolation of studies being done with test systems or test, control, and reference substances known to be biohazardous, including volatile substances, aerosols, radioactive materials, and infectious agents.
(c) Separate areas shall be provided, as appropriate, for the diagnosis, treatment, and control of laboratory test system diseases. These areas shall provide effective isolation for the housing of test systems either known or suspected of being diseased, or of being carriers of disease, from other test systems.
(d) Facilities shall have proper provisions for collection and disposal of contaminated water, soil, or other spent materials. When animals are housed, facilities shall exist for the collection and disposal of all animal waste and refuse or for safe sanitary storage of waste before removal from the testing facility. Disposal facilities shall be so provided and operated as to minimize vermin infestation, odors, disease hazards, and environmental contamination.
(e) Facilities shall have provisions to regulate environmental conditions (e.g., temperature, humidity, photoperiod) as specified in the protocol.
(f) For marine test organisms, an adequate supply of clean sea water or artificial sea water (prepared from deionized or distilled water and sea salt mixture) shall be available. The ranges of composition shall be as specified in the protocol.
(g) For freshwater organisms, an adequate supply of clean water of the appropriate hardness, pH, and temperature, and which is free of contaminants capable of interfering with the study shall be available as specified in the protocol.
(h) For plants, an adequate supply of soil of the appropriate composition, as specified in the protocol, shall be available as needed.
§ 792.45 Test system supply facilities.
(a) There shall be storage areas, as needed, for feed, nutrients, soils, bedding, supplies, and equipment. Storage areas for feed, nutrients, soils, and bedding shall be separated from areas where the test systems are located and shall be protected against infestation or contamination. Perishable supplies shall be preserved by appropriate means.
(b) When appropriate, plant supply facilities shall be provided. These include:
(1) Facilities, as specified in the protocol, for holding, culturing, and maintaining algae and aquatic plants.
(2) Facilities, as specified in the protocol, for plant growth, including but not limited to, greenhouses, growth chambers, light banks, and fields.
(c) When appropriate, facilities for aquatic animal tests shall be provided. These include but are not limited to aquaria, holding tanks, ponds, and ancillary equipment, as specified in the protocol.
§ 792.47 Facilities for handling test, control, and reference substances.
(a) As necessary to prevent contamination or mixups, there shall be separate areas for:
(1) Receipt and storage of the test, control, and reference substances.
(2) Mixing of the test, control, and reference substances with a carrier, e.g., feed.
(3) Storage of the test, control, and reference substance mixtures.
(b) Storage areas for test, control, and/or reference substance and for test, control, and/or reference mixtures shall be separate from areas housing the test systems and shall be adequate to preserve the identity, strength, purity, and stability of the substances and mixtures.
§ 792.49 Laboratory operation areas.
Separate laboratory space and other space shall be provided, as needed, for the performance of the routine and specialized procedures required by studies.
§ 792.51 Specimen and data storage facilities.
Space shall be provided for archives, limited to access by authorized personnel only, for the storage and retrieval of all raw data and specimens from completed studies.
Subpart D – Equipment
§ 792.61 Equipment design.
Equipment used in the generation, measurement, or assessment of data and equipment used for facility environmental control shall be of appropriate design and adequate capacity to function according to the protocol and shall be suitably located for operation, inspection, cleaning, and maintenance.
§ 792.63 Maintenance and calibration of equipment.
(a) Equipment shall be adequately inspected, cleaned, and maintained. Equipment used for the generation, measurement, or assessment of data shall be adequately tested, calibrated, and/or standardized.
(b) The written standard operating procedures required under § 792.81(b)(11) shall set forth in sufficient detail the methods, materials, and schedules to be used in the routine inspection, cleaning, maintenance, testing, calibration, and/or standardization of equipment, and shall specify, when appropriate, remedial action to be taken in the event of failure or malfunction of equipment. The written standard operating procedures shall designate the person responsible for the performance of each operation.
(c) Written records shall be maintained of all inspection, maintenance, testing, calibrating, and/or standardizing operations. These records, containing the date of the operation, shall describe whether the maintenance operations were routine and followed the written standard operating procedures. Written records shall be kept of nonroutine repairs performed on equipment as a result of failure and malfunction. Such records shall document the nature of the defect, how and when the defect was discovered, and any remedial action taken in response to the defect.
Subpart E – Testing Facilities Operation
§ 792.81 Standard operating procedures.
(a) A testing facility shall have standard operating procedures in writing, setting forth study methods that management is satisfied are adequate to insure the quality and integrity of the data generated in the course of a study. All deviations in a study from standard operating procedures shall be authorized by the study director and shall be documented in the raw data. Significant changes in established standard operating procedures shall be properly authorized in writing by management.
(b) Standard operating procedures shall be established for, but not limited to, the following:
(1) Test system room preparation.
(2) Test system care.
(3) Receipt, identification, storage, handling, mixing, and method of sampling of the test, control, and reference substances.
(4) Test system observations.
(5) Laboratory or other tests.
(6) Handling of test systems found moribund or dead during study.
(7) Necropsy of test systems or postmortem examination of test systems.
(8) Collection and identification of specimens.
(9) Histopathology.
(10) Data handling, storage and retrieval.
(11) Maintenance and calibration of equipment.
(12) Transfer, proper placement, and identification of test systems.
(c) Each laboratory or other study area shall have immediately available manuals and standard operating procedures relative to the laboratory or field procedures being performed. Published literature may be used as a supplement to standard operating procedures.
(d) A historical file of standard operating procedures, and all revisions thereof, including the dates of such revisions, shall be maintained.
§ 792.83 Reagents and solutions.
All reagents and solutions in the laboratory areas shall be labeled to indicate identity, titer or concentration, storage requirements, and expiration date. Deteriorated or outdated reagents and solutions shall not be used.
§ 792.90 Animal and other test system care.
(a) There shall be standard operating procedures for the housing, feeding, handling, and care of animals and other test systems.
(b) All newly received test systems from outside sources shall be isolated and their health status or appropriateness for the study shall be evaluated. This evaluation shall be in accordance with acceptable veterinary medical practice or scientific methods.
(c) At the initiation of a study, test systems shall be free of any disease or condition that might interfere with the purpose or conduct of the study. If during the course of the study, the test systems contract such a disease or condition, the diseased test systems should be isolated, if necessary. These test systems may be treated for disease or signs of disease provided that such treatment does not interfere with the study. The diagnosis, authorization of treatment, description of treatment, and each date of treatment shall be documented and shall be retained.
(d) Warm-blooded animals, adult reptiles, and adult terrestrial amphibians used in laboratory procedures that require manipulations and observations over an extended period of time, or in studies that require these test systems to be removed from and returned to their test system-housing units for any reason (e.g., cage cleaning, treatment, etc.), shall receive appropriate identification (e.g., tattoo, color code, ear tag, ear punch, etc.). All information needed to specifically identify each test system within the test system-housing unit shall appear on the outside of that unit. Suckling mammals and juvenile birds are excluded from the requirement of individual identification unless otherwise specified in the protocol.
(e) Except as specified in paragraph (e)(1) of this section, test systems of different species shall be housed in separate rooms when necessary. Test systems of the same species, but used in different studies, should not ordinarily be housed in the same room when inadvertent exposure to test, control, or reference substances or test system mixup could affect the outcome of either study. If such mixed housing is necessary, adequate differentiation by space and identification shall be made.
(1) Plants, invertebrate animals, aquatic vertebrate animals, and organisms that may be used in multispecies tests need not be housed in separate rooms, provided that they are adequately segregated to avoid mixup and cross contamination.
(2) [Reserved]
(f) Cages, racks, pens, enclosures, aquaria, holding tanks, ponds, growth chambers, and other holding, rearing, and breeding areas, and accessory equipment, shall be cleaned and sanitized at appropriate intervals.
(g) Feed, soil, and water used for the test systems shall be analyzed periodically to ensure that contaminants known to be capable of interfering with the study and reasonably expected to be present in such feed, soil, or water are not present at levels above those specified in the protocol. Documentation of such analyses shall be maintained as raw data.
(h) Bedding used in animal cages or pens shall not interfere with the purpose or conduct of the study and shall be changed as often as necessary to keep the animals dry and clean.
(i) If any pest control materials are used, the use shall be documented. Cleaning and pest control materials that interfere with the study shall not be used.
(j) All plant and animal test systems shall be acclimatized to the environmental conditions of the test, prior to their use in a study.
Subpart F – Test, Control, and Reference Substances
§ 792.105 Test, control, and reference substance characterization.
(a) The identity, strength, purity, and composition, or other characteristics which will appropriately define the test, control, or reference substance shall be determined for each batch and shall be documented before its use in a study. Methods of synthesis, fabrication, or derivation of the test, control, or reference substance shall be documented by the sponsor or the testing facility, and such location of documentation shall be specified.
(b) When relevant to the conduct of the study the solubility of each test, control, or reference substance shall be determined by the testing facility or the sponsor before the experimental start date. The stability of the test, control or reference substance shall be determined before the experimental start date or concomitantly according to written standard operating procedures, which provide for periodic analysis of each batch.
(c) Each storage container for a test, control, or reference substance shall be labeled by name, chemical abstracts service number (CAS) or code number, batch number, expiration date, if any, and, where appropriate, storage conditions necessary to maintain the identity, strength, purity, and composition of the test, control, or reference substance. Storage containers shall be assigned to a particular test substance for the duration of the study.
(d) For studies of more than 4 weeks experimental duration, reserve samples from each batch of test, control, and reference substances shall be retained for the period of time provided by § 792.195.
(e) The stability of test, control, and reference substances under storage conditions at the test site shall be known for all studies.
§ 792.107 Test, control, and reference substance handling.
Procedures shall be established for a system for the handling of the test, control, and reference substances to ensure that:
(a) There is proper storage.
(b) Distribution is made in a manner designed to preclude the possibility of contamination, deterioration, or damage.
(c) Proper identification is maintained throughout the distribution process.
(d) The receipt and distribution of each batch is documented. Such documentation shall include the date and quantity of each batch distributed or returned.
§ 792.113 Mixtures of substances with carriers.
(a) For each test, control, or reference substance that is mixed with a carrier, tests by appropriate analytical methods shall be conducted:
(1) To determine the uniformity of the mixture and to determine, periodically, the concentration of the test, control, or reference substance in the mixture.
(2) When relevant to the conduct of the experiment, to determine the solubility of each test, control, or reference substance in the mixture by the testing facility or the sponsor before the experimental start date.
(3) To determine the stability of the test, control or reference substance in the mixture before the experimental start date or concomitantly according to written standard operating procedures, which provide for periodic analysis of each batch.
(b) Where any of the components of the test, control, or reference substance carrier mixture has an expiration date, that date shall be clearly shown on the container. If more than one component has an expiration date, the earliest date shall be shown.
(c) If a vehicle is used to facilitate the mixing of a test substance with a carrier, assurance shall be provided that the vehicle does not interfere with the integrity of the test.
Subpart G – Protocol for and Conduct of A Study
§ 792.120 Protocol.
(a) Each study shall have an approved written protocol that clearly indicates the objectives and all methods for the conduct of the study. The protocol shall contain but shall not necessarily be limited to the following information:
(1) A descriptive title and statement of the purpose of the study.
(2) Identification of the test, control, and reference substance by name, chemical abstracts service (CAS) number or code number.
(3) The name and address of the sponsor and the name and address of the testing facility at which the study is being conducted.
(4) The proposed experimental start and termination dates.
(5) Justification for selection of the test system.
(6) Where applicable, the number, body weight, sex, source of supply, species, strain, substrain, and age of the test system.
(7) The procedure for identification of the test system.
(8) A description of the experimental design, including methods for the control of bias.
(9) Where applicable, a description and/or identification of the diet used in the study as well as solvents, emulsifiers and/or other materials used to solubilize or suspend the test, control, or reference substances before mixing with the carrier. The description shall include specifications for acceptable levels of contaminants that are reasonably expected to be present in the dietary materials and are known to be capable of interfering with the purpose or conduct of the study if present at levels greater than established by the specifications.
(10) The route of administration and the reason for its choice.
(11) Each dosage level, expressed in milligrams per kilogram of body or test system weight or other appropriate units, of the test, control, or reference substance to be administered and the method and frequency of administration.
(12) The type and frequency of tests, analyses, and measurements to be made.
(13) The records to be maintained.
(14) The date of approval of the protocol by the sponsor and the dated signature of the study director.
(15) A statement of the proposed statistical method.
(b) All changes in or revisions of an approved protocol and the reasons therefor shall be documented, signed by the study director, dated, and maintained with the protocol.
§ 792.130 Conduct of a study.
(a) The study shall be conducted in accordance with the protocol.
(b) The test systems shall be monitored in conformity with the protocol.
(c) Specimens shall be identified by test system, study, nature, and date of collection. This information shall be located on the specimen container or shall accompany the specimen in a manner that precludes error in the recording and storage of data.
(d) In animal studies where histopathology is required, records of gross findings for a specimen from postmortem observations shall be available to a pathologist when examining that specimen histopathologically.
(e) All data generated during the conduct of a study, except those that are generated by automated data collection systems, shall be recorded directly, promptly, and legibly in ink. All data entries shall be dated on the day of entry and signed or initialed by the person entering the data. Any change in entries shall be made so as not to obscure the original entry, shall indicate the reason for such change, and shall be dated and signed or identified at the time of the change. In automated data collection systems, the individual responsible for direct data input shall be identified at the time of data input. Any change in automated data entries shall be made so as not to obscure the original entry, shall indicate the reason for change, shall be dated, and the responsible individual shall be identified.
§ 792.135 Physical and chemical characterization studies.
(a) All provisions of the GLPs shall apply to physical and chemical characterization studies designed to determine stability, solubility, octanol water partition coefficient, volatility, and persistence (such as biodegradation, photodegradation, and chemical degradation studies).
(b) The following GLP standards shall not apply to studies designed to determine physical and chemical characteristics of a test, control, or reference substance:
Subparts H-I [Reserved]
Subpart J – Records and Reports
§ 792.185 Reporting of study results.
(a) A final report shall be prepared for each study and shall include, but not necessarily be limited to, the following:
(1) Name and address of the facility performing the study and the dates on which the study was initiated and was completed, terminated, or discontinued.
(2) Objectives and procedures stated in the approved protocol, including any changes in the original protocol.
(3) Statistical methods employed for analyzing the data.
(4) The test, control, and reference substances identified by name, chemical abstracts service (CAS) number or code number, strength, purity, and composition, or other appropriate characteristics.
(5) Stability, and when relevant to the conduct of the study, the solubility of the test, control, and reference substances under the conditions of administration.
(6) A description of the methods used.
(7) A description of the test system used. Where applicable, the final report shall include the number of animals or other test organisms used, sex, body weight range, source of supply, species, strain and substrain, age, and procedure used for identification.
(8) A description of the dosage, dosage regimen, route of administration, and duration.
(9) A description of all circumstances that may have affected the quality or integrity of the data.
(10) The name of the study director, the names of other scientists or professionals and the names of all supervisory personnel, involved in the study.
(11) A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the data, and a statement of the conclusions drawn from the analysis.
(12) The signed and dated reports of each of the individual scientists or other professionals involved in the study, including each person who, at the request or direction of the testing facility or sponsor, conducted an analysis or evaluation of data or specimens from the study after data generation was completed.
(13) The locations where all specimens, raw data, and the final report are to be stored.
(14) The statement prepared and signed by the quality assurance unit as described in § 792.35(b)(7).
(b) The final report shall be signed and dated by the study director.
(c) Corrections or additions to a final report shall be in the form of an amendment by the study director. The amendment shall clearly identify that part of the final report that is being added to or corrected and the reasons for the correction or addition, and shall be signed and dated by the person responsible. Modification of a final report to comply with the submission requirements of EPA does not constitute a correction, addition, or amendment to a final report.
(d) A copy of the final report and of any amendment to it shall be maintained by the sponsor and the test facility.
§ 792.190 Storage and retrieval of records and data.
(a) All raw data, documentation, records, protocols, specimens, and final reports generated as a result of a study shall be retained. Specimens obtained from mutagenicity tests, specimens of soil, water, and plants, and wet specimens of blood, urine, feces, and biological fluids, do not need to be retained after quality assurance verification. Correspondence and other documents relating to interpretation and evaluation of data, other than those documents contained in the final report, also shall be retained.
(b) There shall be archives for orderly storage and expedient retrieval of all raw data, documentation, protocols, specimens, and interim and final reports. Conditions of storage shall minimize deterioration of the documents or specimens in accordance with the requirements for the time period of their retention and the nature of the documents of specimens. A testing facility may contract with commercial archives to provide a repository for all material to be retained. Raw data and specimens may be retained elsewhere provided that the archives have specific reference to those other locations.
(c) An individual shall be identified as responsible for the archives.
(d) Only authorized personnel shall enter the archives.
(e) Material retained or referred to in the archives shall be indexed to permit expedient retrieval.
§ 792.195 Retention of records.
(a) Record retention requirements set forth in this section do not supersede the record retention requirements of any other regulations in this subchapter.
(b)(1) Except as provided in paragraph (c) of this section, documentation records, raw data, and specimens pertaining to a study and required to be retained by this part shall be retained in the archive(s) for a period of at least ten years following the effective date of the applicable final test rule.
(2) In the case of negotiated testing agreements, each agreement will contain a provision that, except as provided in paragraph (c) of this section, documentation records, raw data, and specimens pertaining to a study and required to be retained by this part shall be retained in the archive(s) for a period of at least ten years following the publication date of the acceptance of a negotiated test agreement.
(3) In the case of testing submitted under section 5, except for those items listed in paragraph (c) of this section, documentation records, raw data, and specimens pertaining to a study and required to be retained by this part shall be retained in the archive(s) for a period of at least five years following the date on which the results of the study are submitted to the agency.
(c) Wet specimens, samples of test, control, or reference substances, and specially prepared material which are relatively fragile and differ markedly in stability and quality during storage, shall be retained only as long as the quality of the preparation affords evaluation. Specimens obtained from mutagenicity tests, specimens of soil, water, and plants, and wet specimens of blood, urine, feces, biological fluids, do not need to be retained after quality assurance verification. In no case shall retention be required for longer periods than those set forth in paragraph (b) of this section.
(d) The master schedule sheet, copies of protocols, and records of quality assurance inspections, as required by § 792.35(c) shall be maintained by the quality assurance unit as an easily accessible system of records for the period of time specified in paragraph (b) of this section.
(e) Summaries of training and experience and job descriptions required to be maintained by § 792.29(b) may be retained along with all other testing facility employment records for the length of time specified in paragraph (b) of this section.
(f) Records and reports of the maintenance and calibration and inspection of equipment, as required by § 792.63 (b) and (c), shall be retained for the length of time specified in paragraph (b) of this section.
(g) If a facility conducting testing or an archive contracting facility goes out of business, all raw data, documentation, and other material specified in this section shall be transferred to the archives of the sponsor of the study. The EPA shall be notified in writing of such a transfer.
(h) Specimens, samples, or other non-documentary materials need not be retained after EPA has notified in writing the sponsor or testing facility holding the materials that retention is no longer required by EPA. Such notification normally will be furnished upon request after EPA or FDA has completed an audit of the particular study to which the materials relate and EPA has concluded that the study was conducted in accordance with this part.
(i) Records required by this part may be retained either as original records or as true copies such as photocopies, microfilm, microfiche, or other accurate reproductions of the original records.
PART 795 – PROVISIONAL TEST GUIDELINES
Subpart A [Reserved]
Subpart B – Provisional Chemical Fate Guidelines
§ 795.70 Indirect photolysis screening test: Sunlight photolysis in waters containing dissolved humic substances.
(a) Introduction. (1) Chemicals dissolved in natural waters are subject to two types of photoreaction. In the first case, the chemical of interest absorbs sunlight directly and is transformed to products when unstable excited states of the molecule decompose. In the second case, reaction of dissolved chemical is the result of chemical or electronic excitation transfer from light-absorbing humic species in the natural water. In contrast to direct photolysis, this photoreaction is governed initially by the spectroscopic properties of the natural water.
(2) In general, both indirect and direct processes can proceed simultaneously. Under favorable conditions the measurement of a photoreaction rate constant in sunlight (K
(3) In pure water only, direct photoreaction is possible, although hydrolysis, biotransformation, sorption, and volatilization also can decrease the concentration of a test chemical. By measuring k
(4) Two protocols have been written that measure k
(5) This protocol provides a cost effective test method for measuring k
(6) To correct for variations in solar irradiance during the reaction period, an actinometer is simultaneously insolated. From these data, an indirect photoreaction rate constant is calculated that is applicable to clear-sky, near-surface, conditions in fresh water bodies.
(7) In contrast to k
(8) The value of k
(9) This protocol consists of three separate phases that should be completed in the following order: In Phase 1, SHW is prepared and adjusted; in Phase 2, the test chemical is irradiated in SHW and pure water (PW) to obtain approximate sunlight photoreaction rate constants and to determine whether direct and indirect photoprocesses are important; in Phase 3, the test chemical is again irradiated in PW and SHW. To correct for photobleaching of SHW and also solar irradiance variations, tubes containing SHW and actinometer solutions are exposed simultaneously. From these data k
(b) Phase 1 – Preparation and standardization of synthetic natural water – (1) Approach. (i) Recent studies have demonstrated that natural waters can promote the indirect (or sensitized) photoreaction of dissolved organic chemicals. This reactivity is imparted by dissolved organic material (DOM) in the form of humic substances. These materials absorb sunlight and produce reactive intermediates that include singlet oxygen (
(ii) The indirect photoreactivity of a chemical in a natural water will depend on its response to these reactive intermediates, and possibly others yet unknown, as well as the ability of the water to generate such species. This latter feature will vary from water-to-water in an unpredictable way, judged by the complexity of the situation.
(iii) The approach to standardizing a test for indirect photoreactivity is to use a synthetic humic water (SHW) prepared by water-extracting commercial humic material. This material is inexpensive, and available to any laboratory, in contrast to a specific natural water. The SHW can be diluted to a dissolved organic carbon (DOC) content and uv-visible absorbance typical of most surface fresh waters.
(iv) In recent studies it has been found that the reactivity of SHW mixtures depends on pH, and also the history of sunlight exposure (Mill et al. (1983) under paragraph (f)(11) of this section). The SHW solutions initially photobleach with a time-dependent rate constant. As such, an SHW test system has been designed that is buffered to maintain pH and is pre-aged in sunlight to produce, subsequently, a predictable bleaching behavior.
(v) The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to a standard mixture under defined, reproducible conditions.
(2) Procedure. (i) Twenty grams of Aldrich humic acid are added to a clean 2-liter Pyrex Erlenmeyer flask. The flask is filled with 2 liters of 0.1 percent NaOH solution. A stir bar is added to the flask, the flask is capped, and the solution is stirred for 1 hour at room temperature. At the end of this time the dark brown supernatant is decanted off and either filtered through coarse filter paper or centrifuged and then filtered through 0.4)m microfilter. The pH is adjusted to 7.0 with dilute H
(ii) Pre-aging is accomplished by exposing the concentrated solution in the 2-liter flask to direct sunlight for 4 days in early spring or late fall; 3 days in late spring, summer, or early fall. At this time the absorbance of the solution is measured at 370 nm, and a dilution factor is calculated to decrease the absorbance to 0.50 in a 1 cm path length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the mixture is brought to exact dilution with a precalculated volume of reagent-grade water to give a final absorbance of 0.500 in a 1-cm path length cell at 370 nm. It is tightly capped and refrigerated.
(iii) This mixture is SHW stock solution. Before use it is diluted 10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with an absorbance of 5.00 × 10
(3) Rationale. The foregoing procedure is designed to produce a standard humic-containing solution that is pH controlled, and sufficiently aged that its photobleaching first-order rate constant is not time dependent. It has been demonstrated that after 7 days of winter sunlight exposure, SHW solutions photobleached with a nearly constant rate constant (Mill et al. (1983) under paragraph (f)(11) of this section).
(c) Phase 2 – Screening test – (1) Introduction and purpose. (i) Phase 2 measurements provide approximate solar photolysis rate constants and half-lives of test chemicals in PW and SHW. If the photoreaction rate in SHW is significantly larger than in PW (factor of >2X) then the test chemical is subject to indirect photoreaction and Phase 3 is necessary. Phase 2 data are needed for more accurate Phase 3 measurements, which require parallel solar irradiation of actinometer and test chemical solutions. The actinometer composition is adjusted according to the results of Phase 2 for each chemical, to equalize as much as possible photoreaction rate constants of chemical in SHW and actinometer.
(ii) In Phase 2, sunlight photoreaction rate constants are measured in round tubes containing SHW and then mathematically corrected to a flat water surface geometry. These rate constants are not corrected to clear-sky conditions.
(2) Procedure. (i) Solutions of test chemicals should be prepared using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and reagent-grade (or purer) chemicals.
(ii) This solution should be mixed 9.00:1.00 by volume with PW or SHW stock solution to provide working solutions. In the case of SHW, it gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each working solution should then be transferred to separate 12 × 100 mm quartz tubes with screw tops and tightly sealed with Mininert valves.
(iii) The sample tubes are mounted in a photolysis rack with the tops facing geographically north and inclined 30° from the horizontal. The rack should be placed outdoors over a black background in a location free of shadows and excessive reflection.
(iv) Reaction progress should be measured with an analytical technique that provides a precision of at least ±5 percent. High pressure liquid chromatography (HPLC) or gas chromatograph (GC) have proven to be the most general and precise analytical techniques.
(v) Sample and control solution concentrations are calculated by averaging analytical measurements for each solution. Control solutions should be analyzed at least twice at zero time and at other times to determine whether any loss of chemical in controls or samples has occurred by some adventitious process during the experiment.
(vi) Whenever possible the following procedures should be completed in clear, warm, weather so that solutions will photolyze more quickly and not freeze.
(A) Starting at noon on day zero, expose to sunlight 24 sample tubes mounted on the rack described above. Tape 24 foil-wrapped controls to the bottom of the rack.
(B) Analyze two sample tubes and two unexposed controls in PW and SHW for chemical at 24 hours. Calculate the round tube photolysis rate constants (k
(C) If less than 20 percent conversion occurs in SHW in 1 day, repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16 days, or until 20 percent conversion is reached. Do not extend the experiment past 16 days. If less than 20 percent photoreaction occurs in SHW at the end of 16 days the chemical is “photoinert”. Phase 3 is not applicable.
(D) If more than 80 percent photoreaction occurs at the end of day 1 in SHW, repeat the experiment with eight each of the remaining foil-wrapped PW and SHW controls. Divide these sets into four sample tubes each, leaving four foil-wrapped controls taped to the bottom of the rack.
(1) Expose tubes of chemical in SHW and PW to sunlight starting at 0900 hours and remove one tube and one control at 1, 2, 4, and 8 hours. Analyze all tubes the next day.
(2) Extimate (k
(3) The rate constants (k
(4) Once (k
(vii) Since the rate of photolysis in tubes is faster than the rate in natural water bodies, values of near-surface photolysis rate constants in natural and pure water bodies, k
(3) Criteria for Phase 2. (i) If no loss of chemical is found in dark control solutions compared with the analysis in tubes at zero time (within experimental error), any loss of chemical in sunlight is assumed to be due to photolysis, and the procedure provides a valid estimate of k
(ii) Rate constants determined by the Phase 2 protocol depend upon latitude, season, and weather conditions. Note that (k
(4) Rationale. The Phase 2 protocol is a simple procedure for evaluating direct and indirect sunlight photolysis rate constants of a chemical at a specific time of year and latitude. It provides a rough rate constant for the chemical in SHW that is necessary for Phase 3 testing. By comparison with the direct photoreaction rate constant, it can be seen whether the chemical is subject to indirect photoreaction and whether Phase 3 tests are necessary.
(5) Scope and limitations. (i) Phase 2 testing separates test chemicals into three convenient categories: “Photolabile”, “photoinert”, and those chemicals having sunlight half-lives in round tubes in the range of 1 hour to 50 days. Chemicals in the first two categories fall outside the practical limits of the test, and cannot be used in Phase 3. All other chemicals are suitable for Phase 3 testing.
(ii) The test procedure is simple and inexpensive, but does require that the chemical dissolve in water at sufficient concentrations to be measured by some analytical technique but not have appreciable absorbance in the range 290 to 825 nm. Phase 2 tests should be done during a clear-sky period to obtain the best results. Testing will be less accurate for chemicals with half-lives of less than 1 day because dramatic fluctuations in sunlight intensity can arise from transient weather conditions and the difficulty of assigning equivalent reaction times. Normal diurnal variations also affect the photolysis rate constant. Phase 3 tests should be started as soon as possible after the Phase 2 tests to ensure that the (k
(6) Illustrative Example. (i) Chemical A was dissolved in 0.010 M pH 7.0 buffer. The solution was filtered through a 0.2 )m filter, air saturated, and analyzed. It contained 1.7 × 10
(ii) The SHW solution of A was photolyzed in sealed quartz tubes (12 × 100 mm) in the fall season starting on October 1. At the end of 1 and 2 days, respectively, the concentration of A was found to be 1.13 × 10
(iii) The tube photolysis rate constant of chemical A was calculated from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first time point at day 1 was used because the fraction of A remaining was in the range 20 to 80 percent:
(iv) From this value, k
(v) From measurements in pure water, k
(d) Phase 3 – Indirect photoreaction with actinometer: Calculation of k
(i) The purpose of Phase 3 is to measure k
(ii) In the case (k
(iii) The actinometer used is the p-nitroacetophenone-pyridine (PNAP/PYR) system developed by Dulin and Mill (1982) under paragraph (f)(5) of this section and is used in two EPA test guidelines (USEPA (1984) under paragraphs (f) (14) and (15) of this section). By varying the pyridine concentration, the PNAP photolysis half-life can be adjusted over a range of several hours to several weeks. The starting PNAP concentration is held constant.
(iv) SHW is subject to photobleaching that decreases its ability to promote indirect photolysis based on its ability to absorb sunlight. This effect will be significant when the test period exceeds a few days. To correct for photobleaching, tubes containing SHW are irradiated in action to the other tubes above.
(v) At any time, the loss of test chemical is given by Equation 8 assuming actinometric correction to constant light flux:
(vi) The indirect photolysis rate constant, k
(vii) To evaluate k
Table 1 – Day Averaged Rate Constant (k
1 for Sunlight Absorption by PNAP as a Function of Season and Decadic Latitude
2
| Latitude | Season | |||
|---|---|---|---|---|
| Spring | Summer | Fall | Winter | |
| 20° N | 515 | 551 | 409 | 327 |
| 30° N | 483 | 551 | 333 | 232 |
| 40° N | 431 | 532 | 245 | 139 |
| 50° N | 362 | 496 | 154 | 6 |
1 k
2 For use in Equation 15 under paragraph (d)(2)(i) of this section.
(viii) To obtain k
(ix) Then, (k
(x) Finally, k
(2) Procedure. (i) Using the test chemical photoreaction rate constant in round tubes, (k
(A) The variable k
(B) The variable k
(ii) Once [PYR] is determined, an actinometer solution is prepared by adding 1.00 mL of 1.0 × 10
(iii) The following solutions should be prepared and individually added in 6.00 mL aliquots to 12/100 mm quartz sample tubes; 8 tubes should be filled with each solution:
(A) PNAP/PYR actinometer solution.
(B) Test chemical in pH 7.0, 0.010 M phosphate buffer.
(C) Test chemcial in pH 7.0, 0.010 M phosphate buffer/SHW.
(D) pH 7.0, 0.010 M phosphate buffer/SHW. Four tubes of each set are wrapped in foil and used as controls.
(iv) The tubes are placed in the photolysis rack (Phase 2, Procedure) at 0900 hours on day zero, with the controls taped to the bottom of the rack. One tube of each composition is removed, along with their respective controls, according to a schedule found in Table 2, which categorizes sampling times on the basis of (k
Table 2 – Category and Sampling Procedure for Test and Actinometry Solutions
| Category | k | Sampling procedure |
|---|---|---|
| A | 5.5 J K | Sample at 0, 1, 2, 4, and 8h. |
| B | 0.69>k | Sample at 0, 1, 2, 4, and 8d. |
| C | 0.17>k | Sample at 0, 4, 8, 16, and 32d. |
(v) The tubes containing PNAP, test chemical, and their controls are analyzed for residual concentrations soon after the end of the experiment. PNAP is conveniently analyzed by HPLC, using a 30 cm C
(vi) If controls are well-behaved and show no significant loss of chemical or absorbance change, then k
(vii) According to Equation 12 under paragraph (d)(1)(vii) of this section, plot the quantities Pn(A
(viii) Then, using Equation 13a under paragraph (d)(1)(vii) of this section, determine the slope (S3) by least squares linear regression. Under the assumptions of the protocol, S3 is equal to (k
(ix) From Equation 18
(x) The indirect photoreaction rate constant, k
(xi) The rate constant k
(xii) Then, (k
(xiii) Finally, k
(3) Criteria for Phase 3. As in Phase 2, Phase 3 tests are assumed valid if the dark controls are well behaved and show no significant loss of chemical. In such a case, loss of test chemical in irradiated samples is due to photoreaction.
(4) Rationale. Simultaneous irradiation of a test chemical and actinometer provide a means of evaluating sunlight intensities during the reaction period. Parallel irradiation of SHW solutions allows evaluation of the extent of photobleaching and loss of sensitizing ability of the natural water.
(5) Scope and limitations of Phase 3 protocol. Test chemicals that are classified as having half-lives in SHW in the range of 1 hour to 50 days in Phase 2 listing are suitable for use in Phase 3 testing. Such chemicals have photoreaction half-lives in a range accommodated by the PNAP/PYR actinometry in sunlight and also accommodate the persistence of SHW in sunlight.
(6) Illustrative example. (i) From Phase 2 testing, under paragraph (c)(6)(iii) of this section, chemical A was found to have a photolysis rate constant, (k
(ii) The actinometer solution was made up by adding a volume of pyridine (1.95 mL) calculated from equation 16 under paragraph (d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00 mL of 1.00 × 10
(A) Chemical A (1.53 × 10
(B) Chemical A (1.53 × 10
(C) SHW standard solution diluted with water 0.90 to 1.00 to match solution A.
(D) PNAP/PYR actinometer solution. Ten additional foil-wrapped controls of each mixture were taped to the bottom of the rack.
(iii) The test chemical had been placed in category B, Table 2 under the paragraph (d)(2)(iv) of this section, on the basis of its Phase 2 rate constant under paragraph (c) of this section. Accordingly, two tubes of each irradiated solution and two tubes of each blank solution were removed at 0, 1, 2, 4, and 8 days at 1,200 hours. The averaged analytical results obtained at the end of the experiment are shown in the following Table 3.
Table 3 – Chemical Analytical Results for Illustrative Example, Phase 3
| Day | 10 5[C] SHW, M | 10 5[C] W, M | A SHW | 10 5 [PNAP], M |
|---|---|---|---|---|
| 0 | 1.53 | 1.53 | 0.0500 | 1.00 |
| 1 | 1.03 | 1.40 | 0.0470 | 0.810 |
| 2 | 0.760 | 1.30 | 0.0440 | 0.690 |
| 4 | 0.300 | 1.01 | 0.0370 | 0.380 |
| 8 | 0.130 | 0.800 | 0.0320 | 0.220 |
(A) From these items the functions Pn(C
Table 4 – Photoreaction Function for Illustrative Examples, Phase 3, Derived From Table 3
| Day | Pn(C | Pn(C | 1-(A o | Pn(A o | Pn(C |
|---|---|---|---|---|---|
| 0 | 0 | 0 | 0 | 0 | 0 |
| 1 | 0.396 | 0.0888 | 0.0600 | 0.0618 | 0.211 |
| 2 | 0.700 | 0.163 | 0.120 | 0.128 | 0.371 |
| 4 | 1.629 | 0.415 | 0.260 | 0.301 | 0.968 |
| 8 | 2.465 | 0.648 | 0.360 | 0.446 | 1.514 |
(B) Slope S1 = (k
(C) Slope S2 = (k/k
(D) Using the data in columns 3 and 6 in Table 4 under paragraph (d)(6)(iii)(A) of this section, slope S3 was calculated by regression from Equation 13a under paragraph (d)(1)(viii) of this section and was found to be 0.428 with correlation coefficient equal to 0.99997.
(E) Using Equation 18 under paragraph (d)(2)(ix) of this section, k
(F) The values of S1, S2, and k
(G) The rate constant k
(H) The sum of k
(I) Since k
(e) Data and reporting – (1) Test conditions – (i) Specific analytical and recovery procedures. (A) Provide a detailed description or reference for the analytical procedures used, including the calibration data and precision.
(B) If extraction methods were used to separate the solute from the aqueous solution, provide a description of the extraction method as well as the recovery data.
(ii) Other test conditions. (A) Report the site and latitude where the photolysis experiments were carried out.
(B) Report the dates of photolysis, weather conditions, times of exposure, and the duration of exposure.
(C) If acetonitrile was used to solubilize the test chemical, report the volume percent.
(D) If a significant loss of test chemical occurred in the control solutions for pure water and SHW, indicate the causes and how they were eliminated or minimized.
(2) Test data report – (i) Phase 2 Screening Test under paragraph (c) of this section. (A) Report the initial molar concentration of test chemical, C
(B) Report the molar concentration of test chemical, C
(C) Report the molar concentration of test chemical for each replicate control sample and the mean value for each time point.
(D) Report the values of (k
(E) If small losses of test chemical were observed in SHW and pure water, report a first-order rate constant loss, (k
(F) Report the value of R calculated from Equation 4 under paragraph (c)(2)(vi)(D)(4) of this section.
(G) Report the values of k
(ii) Phase 3 – Indirect photoreaction with actinometer. (A) Report the initial molar concentration of test chemical, C
(B) Report the initial absorbance A
(C) Report the initial molar concentration of PNAP of each replicate and the mean value in the actinometer. Report the concentration of pyridine used in the actinometer which was obtained from Equation 15 under paragraph (d)(2)(i) of this section.
(D) Report the time and date the photolysis experiments were started, the time and date the experiments were completed, and the elapsed photolysis time in days.
(E) For each time point t, report the separate values of the absorbance of the SHW solution, and the mean values.
(F) For each time point for the controls, report the separate values of the molar concentrations of test chemical in pure water and SHW, and the absorbance of the SHW solution, and the mean values.
(G) Tabulate and report the following data: t, [C]
(H) From the data in (G), tabulate and report the following data: t, Pn(C
(I) From the linear regression analysis of the appropriate data in step (H) in Equation 17 under paragraph (d)(2)(vi) of this section, report the slope S1 and the correlation coefficient.
(J) From the linear regression analysis of the appropriate data in step (H) in Equation 12 under paragraph (d)(1)(vii) of this section, report the slope S2 and the correlation coefficient.
(K) From the linear regression analysis of the appropriate data in step (H) in Equation 13a under paragraph (d)(1)(viii) of this section, report the slope S3 and the correlation coefficient.
(L) If loss of chemical was observed during photolysis in pure water and SHW, then report the data Pn(C
(M) Report the value of the actinometer rate constant obtained from Equation 18 under paragraph (d)(2)(ix) of this section.
(N) Report the value of k
(O) Report the value of k
(P) Report the value of (k
(Q) Report the half-life, t
(f) References. For additional background information on this test guideline the following references should be consulted.
(1) Cooper W.J., Zika R.G. “Photochemical formation of hydrogen peroxide in surface and ground waters exposed to sunlight.” Science, 220:711. (1983).
(2) Draper W.M., Crosby D.G. “The photochemical generation of hydrogen peroxide in natural waters.” Archives of Environmental Contamination and Toxicology, 12:121. (1983).
(3) Draper, W.M. and Crosby D.G. “Solar photooxidation of pesticides in dilute hydrogen peroxide.” Journal of Agricultural and Food Chemistry, 32:231. (1984).
(4) Draper W.M., Crosby D.G. “Hydrogen peroxide and hydroxyl radical: Intermediates in indirect photolysis reactions in water.” Journal of Agricultural and Food Chemistry, 29:699. (1981).
(5) Dulin D., Mill T. “Development and evaluation of sunlight actinometers.” Environmental Science and Technology, 6:815. (1982).
(6) Haag H.R., Hoigne J., Gassman E., Braun A.M. “Singlet oxygen in surface waters – Part I; Furfuryl alcohol as a trapping agent.” Chemosphere, 13:631. (1984).
(7) Haag W.R., Hoigne J., Gassman E., Braun A.M. “Singlet oxygen in surface waters – Part II: Quantum yields of its production by some natural humic materials as a function of wavelength.” Chemosphere, 13:641. (1984).
(8) Mill T., Winterle J.S., Fischer A., Tse D., Mabey W.R., Drossman H., Liu A., Davenport J.E. Toxic substances process data generation and protocol development. Work assignment 12, test standard development. “Section 3. Indirect photolysis.” Draft final report. EPA Contract No. 68-03-2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA, and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1984).
(9) Mill T., Mabey W.R., Bomberger D.C., Chou T.W., Hendry D.G., Smith J.H. “Laboratory protocols for evaluating the fate of organic chemicals in air and water. Chapter 3. Photolysis in water. Chapter 4. Oxidation in water.” EPA 600/3-82-022. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA. (1981).
(10) Mill T., Mabey W.R., Winterle J.S., Davenport J.E., Barich V.P., Dulin D.E., Tse D.S., Lee G. “Design and validation of screening and detailed methods for environmental processes. Appendix C. Lower-tier direct photolysis protocol.” Draft final report. EPA Contract No. 68-01-6325. Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1982).
(11) Mill T., Davenport J.E., Winterle J.S., Mabey W.R., Dossman H., Tse D., Liu A. Toxic substances process data generation and protocol development. Work assignment 12. “Appendix B. Upper-tier protocol for direct photolysis in water.” Draft final report. EPA Contract No. 68-03-2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA, and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (July 1983).
(12) Winterle J.S., Mill T. Toxic substances process data generation and protocol development. Work assignment 18. “Indirect photoreaction protocol.” Draft EPA special report. EPA Contract No. 68-03-2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1985).
(13) Mill T., Hendry D.G., Richardson H. “Free radical oxidants in natural waters.” Science, 207:886. (1980).
(14) U.S. Environmental Protection Agency (USEPA), Office of Pollution Prevention and Toxics (OPPT). “Chemical fate test guidelines. Test guideline (CG, CS-6000). Photolysis in aqueous solution.” EPA-560/6-84-003. NTIS publication PB-84-233287. (1984).
(15) USEPA, OPPT. “Chemical fate test guidelines. Test guildeline (CG, CS-6010). Laboratory determination of the direct photolysis reaction quantum yield in aqueous solution and sunlight photolysis.” EPA-560/6-84-003. NTIS publication PB-84-233287. (1984).
(16) Wolff C.J.M., Halmans M.T.H., Van der Heijde H.B. “The formation of singlet oxygen in surface waters.” Chemosphere, 10:59. (1981).
(17) Zepp R.G., Baughman G.L., Schlotzhauer P.F. “Comparison of photochemical behavior of various humic substances in water: I. Sunlight induced reactions of aquatic pollutants photosensitized by humic substances.” Chemosphere, 10:109. (1981).
(18) Zepp R.G., Baughman G.L., Schlozhauer P.F. “Comparison of photochemical behavior of various humic substances in water: II. Photosensitized oxygenations.” Chemosphere, 10:119. (1981).
(19) Zepp R.G., Cline D.M. “Rates of direct photolysis in aquatic environments.” Environmental Science and Technology, 11:359. (1977).
(20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. “Singlet oxygen in natural waters.” Nature, 267:421. (1977).
(21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. “Photosensitized transformations involving electronic energy transfer in natural waters: role of humic substances.” Environmental Science and Technology, 19:74. (1985).
Subpart C – Provisional Environmental Effects Guidelines
§ 795.120 Gammarid acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data on the acute toxicity of chemical substances and mixtures subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003 (15 U.S.C. 2601 et seq.)). This guideline describes a test to develop data on the acute toxicity of chemicals to gammarids. The United States Environmental Protection Agency (EPA) will use data from this test in assessing the hazard of a chemical to aquatic organisms.
(b) Definitions. The definitions in section 3 of TSCA and in part 792 of this chapter, Good Laboratory Practice Standards, apply to this test guideline. The following definitions also apply to this guideline:
Death means the lack of reaction of a test organism to gentle prodding.
Flow-through means a continuous or an intermittent passage of test solution or dilution water through a test chamber or a holding or acclimation tank, with no recycling.
LC50 means the median lethal concentration, i.e., that concentration of a chemical in air or water killing 50 percent of the test batch of organisms within a particular period of exposure (which shall be stated).
Loading means the ratio of the biomass of gammarids (grams, wet weight) to the volume (liters) of test solution in either a test chamber or passing through it in a 24-hour period.
Solvent means a substance (e.g., acetone) which is combined with the test substance to facilitate introduction of the test substance into the dilution water.
Static system means a test chamber in which the test solution is not renewed during the period of the test.
(c) Test procedures – (1) Summary of the test. In preparation for the test, test chambers are filled with appropriate volumes of dilution water. If a flow-through test is performed, the flow of dilution water through each chamber is adjusted to the rate desired. In a static test, the test substance is introduced into each test chamber. In a flow-through test, the rate in which the test substance is added is adjusted to establish and maintain the desired concentration of test substance in each test chamber. The test is started by randomly introducing gammarids, which have been acclimated to the test conditions, into the test chambers. Gammarids in the test chambers are observed periodically during the test; the dead gammarids are removed and the findings recorded. Dissolved oxygen concentration, pH, temperature, and the concentration of test substance in test chambers are measured at specified intervals. Data collected during the test are used to develop concentration – response curves and LC50 values for the test substance.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted to establish test substance concentrations to be used for the definitive test.
(ii) The gammarids shall be exposed to a wide-range of concentrations of the test substance (e.g., 1, 10, 100 mg/1, etc.), usually under static conditions.
(iii) A minimum of five gammarids should be exposed to each concentration of test substance for a period of 96 hours. The exposure period may be shortened if data suitable for determining concentrations in the definitive test can be obtained in less time. Nominal concentrations of the test substance may be acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to determine the 24, 48, 72, and 96 – hour LC50 values and the concentration-response curves.
(ii) A minimum of 20 gammarids per concentration shall be exposed to five or more concentrations of the test substance chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/L). The range and number of concentrations to which the organisms are exposed shall be such that in 96 hours there is at least one concentration resulting in mortality greater than 50 and less than 100 percent, and one concentration causing greater than zero and less than 50 percent mortality. An equal number of gammarids may be placed in two or more replicate test chambers. Solvents should be avoided, if possible. If solvents have to be used, a solvent control, as well as a dilution control, shall be tested at the highest solvent concentration employed in the treatments. The solvent should not be toxic or have an effect on the toxicity of the test substance. The concentration of solvent should not exceed 0.1 ml/L.
(iii) Every test shall include a concurrent control using gammarids from the same population or culture container. The control group shall be exposed to the same dilution water, conditions and procedures, except that none of the test substance shall be is added to the chamber.
(iv) The dissolved oxygen concentration, temperature and pH of the test solution shall be measured at the beginning of the test and at 24, 48, 72 and 96 hours in at least one replicate each of the control, and the highest, lowest and middle test concentrations.
(v) The test duration is 96 hours. The test is unacceptable if more than 10 percent of the control organisms die during the test.
(vi) In addition to death, any abnormal behavior or appearance shall also be reported.
(vii) Gammarids shall be randomly assigned to the test chambers. Test chambers shall be positioned within the testing area in a random manner or in a way in which appropriate statistical analyses can be used to determine whether there is any variation due to placement.
(viii) Gammarids shall be introduced into the test chambers after the test substance has been added.
(ix) Observations on compound solubility shall be recorded. The investigator should record the appearance of surface slicks, precipitates, or material adhering to the sides of the test chambers.
(5) [Reserved]
(6) Analytical measurements – (i) Water quality analysis. The hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and particulate matter of the dilution water shall be measured at the beginning of each definitive test.
(ii) Collection of samples for measurement of test substance. Each sample to be analyzed for the test substance concentrations shall be taken at a location midway between the top, bottom, and sides of the test chamber. Samples should not include any surface scum or material dislodged from the bottom or sides. Samples shall be analyzed immediately or handled and stored in a manner which minimizes loss of test substance through microbial degradation, photogradation, chemical reaction, volatilization, or sorption.
(iii) Measurement of test substance. (A) For static tests, the concentration of dissolved test substance (that which passes through a 0.45 micron filter) shall be measured in each test chamber at least at the beginning (zero-hour, before gammarids are added) and at the end of the test. During flow-through tests, the concentration of dissolved test substance shall be measured in each test chamber at least at 0 and 96-hours and in at least one chamber whenever a malfunction of the test substance delivery system is observed.
(B) The analytical methods used to measure the amount of test substance in a sample shall be validated before beginning the test. This involves adding a known amount of the test substance to each of three water samples taken from a chamber containing dilution water and the same number of gammarids as are placed in each test chamber. The nominal concentrations of the test substance in these samples should span the concentration range to be used in the test. Validation of the analytical method should be performed on at least two separate days prior to starting the test.
(C) An analytical method is not acceptable if likely degradation products of the test substance give positive or negative interferences, unless it is shown that such degradation products are not present in the test chambers during the test.
(D) Among replicate test chambers, the measured concentrations shall not vary more than 20 percent. The measured concentration of the test substance in any chamber during the test shall not vary more than plus or minus 30 percent from the measured concentration in that chamber at zero time.
(E) The mean measured concentration of dissolved test substance shall be used to calculate all LC50’s and to plot all concentration-response curves.
(d) Test conditions for definitive test – (1) Test species – (i) Selection. (A) The amphipods, Gammarus fasciatus, G. pseudolimnaeus, and G. lacustris are specified for this test.
(B) Gammarids can be cultured in the laboratory or collected from natural sources. If collected, they must be held in the laboratory for at least 14 days prior to testing.
(C) Gammarids used in a particular test shall be of similar age and/or size and from the same source or culture population.
(ii) Acclimation. If the holding water is from the same source as the dilution water, acclimation to the dilution water shall be done gradually over a 48-hour period. The gammarids then shall be held at least 7 days in the dilution water prior to testing. Any changes in water temperature should not exceed 2 °C per day. Gammarids should be held for a minimum of 7 days at the test temperature prior to testing.
(iii) Care and handling. Gammarids shall be cultured in dilution water under similar environmental conditions to those used in the test. Organisms shall be handled as little as possible. When handling is necessary it should be done as gently, carefully and quickly as possible. During culturing and acclimation, gammarids shall be observed carefully for signs of stress and mortality. Dead and abnormal individuals shall be discarded.
(iv) Feeding. The organisms shall not be fed during testing. During culturing, holding, and acclimation, a sufficient quantity of deciduous leaves, such as maple, aspen, or birch, should be placed in the culture and holding containers to cover the bottom with several layers. These leaves should be aged for at least 30 days in a flow-through system before putting them in aquaria. As these leaves are eaten, more aged leaves should be added. Pelleted fish food may also be added.
(2) Facilities – (i) Apparatus – (A) Facilities needed to perform this test include:
(1) Containers for culturing, acclimating and testing gammarids;
(2) Containers for aging leaves under flow-through conditions;
(3) A mechanism for controlling and maintaining the water temperature during the culturing, acclimation and test periods;
(4) Apparatus for straining particulate matter, removing gas bubbles, or aerating the dilution water, as necessary; and
(5) An apparatus for providing a 16-hour light and 8-hour dark photoperiod with a 15- to 30-minute transition period.
(B) Facilities should be well ventilated and free of fumes and disturbances that may affect the test organism.
(C) Test chambers shall be covered loosely to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions.
(ii) Construction materials. Construction materials and equipment that may contact the stock solution, test solution or dilution water should not contain substances that can be leached or dissolved into aqueous solutions in quantities that can alter the test results. Materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test substances. Glass, stainless steel, and perfluorocarbon plastic should be used wherever possible. Concrete, fiberglass, or plastic (e.g., PVC) may be used for holding tanks, acclimation tanks, and water supply systems, but they should be aged prior to use. Rubber, coopper, brass, galvanized metal, and lead should not come in contact with the dilution water, stock solution, or test solution.
(iii) Test substance delivery system. In flow-through tests, diluters, metering pump systems or other suitable devices shall be used to deliver the test substance to the test chambers. The system used shall be calibrated before each test. The general operation of the test substance delivery system shall be checked twice daily during a test. The 24-hour flow shall be equal to at least five times the volume of the test chamber. During a test, the flow rates should not vary more than 10 percent from one test chamber to another.
(iv) Test chambers. Test chambers shall contain at least one liter of test solution. Test chambers made of stainless steel should be welded, not soldered. Test chambers made of glass should be glued using clear silicone adhesive. As little adhesive as possible should be left exposed in the interior of the chamber. A substrate, such as a bent piece of stainless steel screen, should be placed on the bottom of each test chamber to provide cover for the gammarids.
(v) Cleaning of test system. Test substance delivery systems and test chambers should be cleaned before each test. They should be washed with detergent and then rinsed sequentially with clean water, pesticide-free acetone, clean water, and 5-percent nitric acid, followed by two or more changes of dilution water.
(vi) Dilution water. (A) Clean surface or ground water, reconstituted water, or dechlorinated tap water is acceptable as dilution water if gammarids will survive in it for the duration of the culturing, acclimating, and testing periods without showing signs of strees. The quality of the dilution water should be constant enough that the month-to-month variation in hardness, acidity, alkalinity, conductivity, TOC or COD, and particulate matter is not more than 10 percent. The pH should be constant within 0.4 unit. In addition, the dilution water should meet the following specifications measured at least twice a year:
| Substance | Maximum concentration |
|---|---|
| Particulate matter | 20 mg/L |
| Total organic carbon (TOC) or | 2 mg/L |
| chemical oxygen demand (COD) | 5 mg/L |
| Boron, fluoride | 100 ug/L |
| Un-ionized ammonia | 1 ug/L |
| Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, zinc | 1 ug/L |
| Residual chlorine | 3 ug/L |
| Cadmium, mercury, silver | 100 ng/L |
| Total organophosphorus pesticides | 50 ng/L |
| Total organochlorine pesticides plus: | |
| polychlorinated biphenyls (PCBs) or | 50 ng/L |
| organic chlorine | 25 ng/L |
(B) If the dilution water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) shall be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilled water. Glass-distilled or carbon-filtered deionized water with a conductivity less than 1 micromho/cm is acceptable as the diluent for making reconstituted water.
(C) The concentration of dissolved oxygen in the dilution water shall be between 90 and 100 percent saturation. If necessary, the dilution water can be aerated before the addition of the test substance. All reconstituted water should be aerated before use.
(3) Test parameters. Environmental parameters during the test shall be maintained as specified below:
(i) Water temperature of 18 ±1 °C.
(ii) Dissolved oxygen concentration between 60 and 105 percent saturation.
(iii) The number of gammarids placed in a test chamber shall not be so great as to affect the results of the test. Ten gammarids per liter is the recommended level of loading for the static test. Loading requirements for the flow-through test will vary depending on the flow rate of dilution water. The loading should not cause the dissolved oxygen concentration to fall below the recommended levels.
(iv) Photoperiod of 16 hours light and 8 hours darkness.
(e) Reporting. The sponsor shall submit to the EPA all data developed by the test that are suggestive or predictive of toxicity. In addition, the test report shall include, but not necessarily be limited to, the following information:
(1) Name and address of the facility performing the study and the dates on which the study was initiated and completed.
(2) Objectives and procedures stated in the approved protocol, including any changes in the original protocol.
(3) Statistical methods employed for analyzing the data.
(4) The test substance identified by name, Chemical Abstracts (CAS) number or code number, source, lot or batch number, strength, purity, and composition, or other appropriate characteristics.
(5) Stability of the test substance under the conditions of the test.
(6) A description of the methods used, including:
(i) The source of the dilution water, its chemical characteristics (e.g., hardness, pH, etc.) and a description of any pretreatment.
(ii) A description of the test substance delivery system, test chambers, the depth and volume of solution in the chamber, the way the test was begun (e.g., test substance addition), the loading, the lighting, and the flow rate.
(iii) Frequency and methods of measurements and observations.
(7) The scientific name, weight, length, source, and history of the organisms used, and the acclimation procedures and food used.
(8) The concentrations tested, the number of gammarids and replicates per test concentration. The reported results should include:
(i) The results of dissolved oxygen, pH and temperature measurements.
(ii) If solvents are used, the name and source of the solvent, the nominal concentration of the test substance in the stock solution, the highest solvent concentration in the test solution and a description of the solubility determination in water and solvents.
(iii) The measured concentration of the test substance in each test chamber just before the start of the test and at all subsequent sampling periods.
(iv) In each test chamber at each observation period, the number of dead and live test organisms, the percentage of organisms that died, and the number of test organisms that showed any abnormal effects in each test chamber at each observation period.
(v) The 48, 72 and 96-hour LC50’s and their 95 percent confidence limits. When sufficient data have been generated, the 24-hour LC50 value also. These calculations should be made using the mean measured test substance concentrations.
(vi) The observed no-effect concentration (the highest concentration tested at which there were no mortalities or abnormal behavioral or physiological effects), if any.
(vii) Methods and data for all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks.
(9) A description of all circumstances that may have affected the quality or integrity of the data.
(10) The names of the sponsor, study director, principal investigator, names of other scientists or professionals, and the names of all supervisory personnel involved in the study.
(11) A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the data, and a statement of the conclusions drawn from the analysis. Results of the analysis of data should include the calculated LC50 value, 95 percent confidence limits, slope of the transformed concentration-response line, and the results of a goodness-of-fit test (e.g., chi-square test).
(12) The signed and dated reports prepared by any individual scientist or other professional involved in the study, including each person who, at the request or direction of the testing facility or sponsor, conducted an analysis or evaluation of data or specimens from the study after data generation was completed.
(13) The locations where all specimens, raw data, and the final report are stored.
(14) The statement prepared and signed by the quality assurance unit.
Subpart D – Provisional Health Effects Guidelines
§ 795.225 Dermal pharmacokinetics of DGBE and DGBA.
(a) Purpose. The purpose of these studies is to determine:
(1) The absorption of diethylene glycol butyl ether (DGBE) after administration by the dermal route.
(2) The biotransformation of DGBE administered dermally.
(3) The dermal absorption of DGBE and diethylene glycol butyl ether acetate (DGBA).
(b) Test procedures – (1) Animal selection – (i) Species. The species utilized for investigating DGBE and DGBA shall be the rat, a species for which historical data on the toxicity and carcinogenicity of many compounds are available and which is used extensively in percutaneous absorption studies.
(ii) Animals. Adult female Sprague Dawley rats shall be used. The rats shall be 7 to 8 weeks old and weigh 180 to 220 grams. Prior to testing, the animals shall be selected at random for each group. Animals showing signs of ill health shall not be used.
(iii) Animal care. (A) The animals should be housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms should be maintained at a temperature of 25 ±2 °C and humidity of 50 ±10 percent with a 12-hour light/dark cycle per day. The rats should be isolated for at least 7 days prior to use.
(B) During the acclimatization period, the rats should be housed in cages on hardwood chip bedding. All animals shall be provided with conventional laboratory diets and water ad libitum.
(2) Administration of DGBE and DGBA – (i) Test substances. These studies require the use of
(ii) Dosage and treatment. (A) Two doses of DGBA shall be used in the study, a “low” dose and a “high” dose. Three doses of DGBE shall be used in the study, a neat “low” dose, an aqueous “low” dose, and neat “high” dose. When administered dermally, the “high” dose level should ideally induce some overt toxicity such as weight loss. The “low” dose level should correspond to a no observed effect level.
(B) For dermal treatment, the doses shall be applied in a volume adequate to deliver the prescribed doses. The backs of the rats should be lightly shaved with an electric clipper shortly before treatment. The dose shall be applied with a micropipette on a specific area (for example, 2 cm
(iii) Washing efficiency study. Before initiation of the dermal absorption studies described in paragraph (b)(2)(iv)(A) of this section, an initial washing efficiency experiment shall be performed to assess the extent of removal of the applied DGBE and DGBA by washing with soap and water. Groups of four rats should be lightly anesthetized with sodium pentobarbital. These animals shall then be treated with dermal doses of test substance at the low dose level. Soon after application (5 to 10 minutes) the treated animals shall be washed with soap and water then housed in individual metabolism cages for excreta collection. Urine and feces shall be collected at 8, 24, and 48 hours following dosing. Collection of excreta shall continue every 24 hours if a significant amounts of DGBE, DGBA, or metabolites continue to be eliminated.
(iv) Determination of absorption, biotransformation, and excretion. (A) Eight animals shall be dosed once dermally with the low dose of
(B) Eight animals shall be dosed once dermally with the high dose of
(C) Eight animals shall be dosed once dermally with the low dose of
(D) Eight animals shall be dosed once dermally with the high dose of
(E) The high and low doses of
(3) Observation of animals – (i) Urinary and fecal excretion. The quantities of total
(ii) Biotransformation after dermal dosing. Appropriate qualitative and quantitative methods shall be used to assay urine specimens collected from rats dosed with DGBE as specified in paragraph (b)(2)(iv) of this section. Any metabolite which comprises greater than 10 percent of the dose shall be identified.
(c) Data and reporting – (1) Treatment of results. Data shall be summarized in tabular form.
(2) Evaluation of results. All observed results, quantitative or incidental, shall be evaluated by an appropriate statistical method.
(3) Test report. In addition to the reporting requirements as specified in the TSCA Good Laboratory Practice Standards, in part 792, subpart J of this chapter, the following specific information shall be reported:
(i) Species, strain, and supplier of laboratory animals.
(ii) Information on the degree (i.e., specific activity for a radiolabel) and sites of labeling of the test substances.
(iii) A full description of the sensitivity and precision of all procedures used to produce the data.
(iv) Relative percent absorption by the dermal route for rats administered low and high doses of
(v) Quantity of isotope, together with percent recovery of the administered dose, in feces and urine.
(vi) Biotransformation pathways and quantities of DGBE and metabolites in urine collected after administering single high and low dermal doses to rats.
§ 795.228 Oral/dermal pharmacokinetics.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of a chemical substance or mixture (“test substance”) are similar after oral and dermal administration.
(2) Determine bioavailability of a test substance after oral and dermal administration.
(3) Examine the effects of repeated dosing on the pharmacokinetics and metabolism of the test substance.
(b) Definitions. (1) Bioavailability refers to the rate and relative amount of administered test substance which reaches the systemic circulation.
(2) Metabolism means the study of the sum of the processes by which a particular substance is handled in the body and includes absorption, tissue distribution, biotransformation, and excretion.
(3) Percent absorption means 100 times the ratio between total excretion of radioactivity following oral or dermal administration and total excretion following intravenous administration of test substance.
(4) Pharmacokinetics means the study of the rates of absorption, tissue distribution, biotransformation, and excretion.
(c) Test procedures – (1) Animal selection – (i) Species. The rat shall be used for pharmacokinetics testing because it has been used extensively for metabolic and toxicological studies. For dermal bioavailability studies, the rat and the mini-pig shall be used.
(ii) Test animals. For pharmacokinetics testing and dermal studies, adult male and female Sprague-Dawley rats, 7 to 9 weeks of age, shall be used. For dermal studies, young adult mini-pigs shall also be used. The animals should be purchased from a reputable dealer and shall be identified upon arrival at the testing laboratory. The animals shall be selected at random for the test groups and any animal showing signs of ill health shall not be used. In all studies, unless otherwise specified, each test group shall contain at least 4 animals of each sex for a total of at least 8 animals.
(iii) Animal care. (A) The animals shall be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 24 ±2 °C and humidity of 50 ±20 percent with a 12-hour light/dark cycle per day. The animals shall be kept in a quarantine facility for at least 7 days prior to use and shall be acclimated to the experimental environment for a minimum of 48 hours prior to administration of the test substance.
(B) During the acclimatization period, the animals shall be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum. The mini-pig diet shall be supplemented with adequate amounts of ascorbic acid in the drinking water.
(2) Administration of test substance – (i) Test substance. The use of a radioactive test substance is required for all studies. Ideally, the purity, radioactive and nonradioactive, is greater than 99 percent. The radioactive and nonradioactive test substances shall be chromatographed separately and together to establish purity and identity. If the purity is less than 99 percent or if the chromatograms differ significantly, EPA should be consulted.
(ii) Dosage and treatment – (A) Intravenous. The low dose of test substance, in an appropriate vehicle, shall be administered intravenously to groups of rats and mini-pigs of each sex. If feasible, the same low dose should be used for intravenous, oral, and dermal studies.
(B) Oral. Two doses of text substance shall be used in the oral study, a low dose and a high dose. The high dose should ideally induce some overt toxicity, such as weight loss. The low dose should correspond to a no-observed effect level. The oral dosing shall be accomplished by gavage or by administering the encapsulated test substance. If feasible, the same high and low doses should be used for oral and dermal studies.
(C) Dermal. (1) Dermal treatment. For dermal treatment, two doses, comparable to the low and high oral doses, shall be dissolved in a suitable vehicle and applied in volumes adequate to deliver comparable doses. The backs of the animals should be lightly shaved with an electric clipper 24 hours before treatment. The test substance shall be applied to the intact shaven skin (approximately 2 cm
(2) Washing efficacy study. Before initiation of the dermal absorption studies, an initial washing efficacy experiment shall be conducted to assess the removal of the applied low dose of the test substance by washing the exposed skin area with soap and water and an appropriate organic solvent. The low dose shall be applied to 4 rats and 4 mini-pigs in accordance with paragraph (c)(2)(ii)(C)(1) of this section. After application (5 to 10 minutes), the treated areas of 2 rats and 2 mini-pigs shall be washed with soap and water and the treated areas of the remaining rats and pigs shall be washed with an appropriate solvent. The amounts of test substance recovered in the washings shall be determined to assess efficacy of its removal by washing.
(iii) Dosing and sampling schedule – (A) Rat studies. After administration of the test substance, each rat shall be placed in a metabolic unit to facilitate collection of excreta. For the dermal studies, excreta from the rats shall also be collected during the 6 hour exposure periods. At the end of each collection period, the metabolic units shall be cleaned to recover any excreta that might adhere to them. All studies, except the repeated dosing study, shall be terminated at 7 days or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first.
(1) Intravenous study. Group A shall be dosed once intravenously at the low dose of test substance.
(2) Oral study. (i) Group B shall be dosed once per os with the low dose of test substance.
(ii) Group C shall be dosed once per os with the high dose of test substance.
(3) Dermal studies. Unless precluded by corrosivity, the test substance shall be applied and kept on the skin for a minimum of 6 hours. At the time of removal of the porous covering, the treated area shall be washed with an appropriate solvent to remove any test substance that may be on the skin surface. Both the covering and the washing shall be assayed to recover residual radioactivity. At the termination of the studies, each animal shall be sacrificed and the exposed skin area removed. An appropriate section of the skin shall be solubilized and assayed for radio-activity to ascertain if the skin acts as a reservoir for the test substance. Studies on the dermal absorption of corrosive test substances should be discussed with EPA prior to initiation.
(i) Group D shall be dosed once dermally with the low dose of test compound.
(ii) Group E shall be dosed once dermally with the high dose of the test substance.
(4) Repeated dosing study. Group F shall receive a series of single daily oral low doses of nonradioactive test substance over a period of at least 7 days. Twenty-four hours after the last nonradioactive dose, a single oral low dose of radioactive test substance shall be administered. Following dosing with the radioactive substance, the rats shall be placed in individual metabolic units as described in paragraph (c)(2)(iii) of this section. The study shall be terminated at 7 days after the last dose, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first.
(B) Mini-Pig studies. For all mini-pig studies, the test groups shall consist of four young adult animals. After administration of the test substance, each mini-pig shall be kept in a metabolic unit to facilitate collection of excreta. At the end of each collection period, the metabolic units are to be cleaned to recover any excreta that might adhere to them. All studies shall be terminated at 7 days, or after at least 90 percent of the radio-activity has been recovered in the excreta, whichever occurs first.
(1) Intravenous study. Group G is to be dosed once intravenously at the low dose of the test substance.
(2) Dermal studies. Following the experimental guidance described in (c)(2)(iii)(A)(3) of this section:
(i) Group H shall be dosed once dermally with the low dose of test substance.
(ii) Group I shall be dosed once dermally with the high dose of the test substance.
(3) Types of studies – (i) Pharmacokinetics studies – (A) Rat studies. Groups A through F shall be used to determine the kinetics of absorption of the test substance. In the group administered the test substance by intravenous routes, (i.e., Group A), the concentration of radioactivity in blood and excreta shall be measured following administration. In groups administered the test substance by the oral and dermal route (i.e., Groups B, C, D, E and F), the concentration of radioactivity in blood and excreta shall be measured at selected time intervals during and following the exposure period.
(B) Mini-Pig studies. Groups G, H, and I shall be used to determine the extent of dermal absorption of the test substance. The amount of radioactivity in excreta shall be determined at selected time intervals.
(ii) Metabolism studies – Rat studies. Groups A through F shall be used to determine the metabolism of the test substance. Urine, feces, and expired air shall be collected for identification and quantification of the test substance and metabolites.
(4) Measurements – (i) Pharmacokinetics. Four animals from each group shall be used for these purposes.
(A) Rat studies – (1) Bioavailability. The levels of radioactivity shall be determined in whole blood, blood plasma or blood serum at 15 and 30 minutes and at 1, 2, 8, 24, 48, and 96 hours after initiation of dosing.
(2) Extent of absorption. The total quantities of radioactivity shall be determined for excerta collected daily for 7 days or until at least 90 percent of the radioactivity has been recovered in the excreta.
(3) Excretion. The quantities of radioactivity eliminated in the urine, feces, and expired air shall be determined separately at appropriate time intervals. The collection of carbon dioxide may be discontinued when less than one percent of the dose is found to be exhaled as radioactive carbon dioxide in 24 hours.
(4) Tissue distribution. At the termination of each study, the quantities of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin, and residual carcass of each animal shall be determined.
(5) Changes in pharmacokinetics. Results of pharmacokinetics measurements (i.e., bioavailability and extent of absorption, tissue distribution, and excretion) obtained in rats receiving the single low oral dose of the test substance (Groups B and C) shall be compared to the corresponding results obtained in rats receiving repeated oral doses of the test substance (Group F).
(B) Mini-Pig studies – Extent of absorption. The total quantities of radioactivity shall be determined for excreta daily for 7 days or until at least 90 percent of the test substance has been excreted.
(ii) Metabolism. Four animals from each group shall be used for these purposes.
(A) Rat studies – (1) Biotransformation. Appropriate qualitative and quantitative methods shall be used to assay urine, feces, and expired air collected from rats. Efforts shall be made to identify any metabolite which comprises 5 percent or more of the administered dose and the major radioactive components of blood.
(2) Changes in biotransformation. Appropriate qualitative and quantitative assay methodology shall be used to compare the composition of radioactive compounds in excreta from rats receiving a single oral dose (Groups B and C) with those in the excreta from rats receiving repeated oral doses (Group H).
(d) Data and reporting. The final test report shall include the following:
(1) Presentation of results. Numerical data shall be summarized in tabular form. Pharmacokinetic data shall also be presented in graphical form. Qualitative observations shall also be reported.
(2) Evaluation of results. All quantitative results shall be evaluated by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as specified in 40 CFR part 792, the following specific information shall be reported:
(i) Species and strains of laboratory animals.
(ii) Chemical characterization of the test substance, including:
(A) For the radioactive test substances, information on the site(s) and degree of radiolabeling, including type of label, specific activity, chemical purity, and radiochemical purity.
(B) For the nonradioactive compound, information on chemical purity.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy of all procedures used to generate the data.
(iv) Percent of absorption of test substance after oral and dermal exposures to rats and dermal exposure to mini-pigs.
(v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. In dermal studies on rats and mini-pigs, include recovery data for skin, skin washings, and residual radioactivity in the covering as well as results of the washing efficacy study.
(vi) Tissue distribution reported as quantity of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin and in residual carcass of rats.
(vii) Materials balance developed from each study involving the assay of body tissues and excreta.
(viii) Biotransformation pathways and quantities of test substance and metabolites in excreta collected after administering single high and low doses to rats.
(ix) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering repeated low doses to rats.
(x) Pharmacokinetics model(s) developed from the experimental data.
§ 795.231 Pharmacokinetics of isopropanal.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of the “test substance” are similar after oral and inhalation administration.
(2) Determine bioavailability of the test substance after oral and inhalation administration.
(3) Examine the effects of repeated dosing on the pharmacokinetics and metabolism of the test substance.
(b) Definitions. (1) “Bioavailability” refers to the rate and relative amount of administered test substance which reaches the systemic circulation.
(2) “Metabolism” means the study of the sum of the processes by which a particular substance is handled in the body, and includes absorption, tissue distribution, biotransformation, and excretion.
(3) “Pharmacokinetics” means the study of the rates of absorption, tissue distribution, biotransformation, and excretion.
(c) Test procedures – (1) Animal selection – (i) Species. The rat shall be used because it has been used extensively for metabolic and toxicological studies.
(ii) Test animals. For pharmacokinetics testing, adult male and female rats (Fischer 344 or strain used for major toxicity testing), 7 to 9 weeks of age, shall be used. The animals should be purchased from a reputable dealer and shall be identified upon arrival at the testing laboratory. The animals shall be selected at random for the testing groups and any animal showing signs of ill health shall not be used. In all studies, unless otherwise specified, each test group shall contain at least four animals of each sex for a total of at least eight animals.
(iii) Animal care. (A) Animal care and housing should be in accordance with DHEW Publication No. (NIH)-85-23, 1985, entitled “Guidelines for the Care and Use of Laboratory Animals.”
(B) The animals should be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 22 ±2 °C and humidity of 50 ±20 percent with a 12-hour light/dark cycle per day. The animals shall be kept in a quarantine facility for at least 7 days prior to use and shall be acclimated to the experimental environment for a minimum of 48 hours prior to treatment.
(C) During the acclimatization period, the animals should be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum.
(2) Administration of test substance – (i) Test substance. The use of radioactive test substance is required for all materials balance and metabolite identification requirements of the study. Ideally, the purity of both radioactive and nonradioactive test substance should be greater than 99 percent. The radioactive and nonradioactive substances shall be chromatographed separately and together to establish purity and identity. If the purity is less than 99 percent or if the chromatograms differ significantly, EPA should be consulted.
(ii) Dosage and treatment – (A) Intravenous. The low dose of test substance, in an appropriate vehicle, shall be administered intravenously to four rats of each sex.
(B) Oral. Two doses of test substance shall be used in the oral portion of the study, a low dose and a high dose. The high dose should ideally induce some overt toxicity, such as weight loss. The low dose level should correspond to a no-observed effect level. The oral dosing shall be accomplished by gavage or by administering an encapsulated test substance. If feasible, the same high and low doses should be used for oral and dermal studies.
(C) Inhalation. Two concentrations of the test substance shall be used in this portion of the study, a low concentration and a high concentration. The high concentration should ideally induce some overt toxicity, while the low concentration should correspond to a no observed level. Inhalation treatment should be conducted using a “nose-cone” or “head only” apparatus to prevent ingestion of the test substance through “grooming”.
(iii) Dosing and sampling schedule. After administration of the test substance, each rat shall be placed in a separate metabolic unit to facilitate collection of excreta. For the inhalation studies, excreta from the rats shall also be collected during the exposure periods. At the end of each collection period, the metabolic units shall be cleaned to recover any excreta that might adhere to the cages. All studies, except the repeated dose study, shall be terminated at 7 days, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first.
(A) Intravenous study. Group A shall be dosed once intravenousely at the low dose of test substance.
(B) Oral studies. (1) Group B shall be dosed once per os with the low dose of the test substance.
(2) Group C shall be dosed once per os with the high dose of the test substance.
(C) Inhalation studies. A single 6-hour exposure period shall be used for each group.
(1) Group D shall be exposed to a mixture of the test substance in air at the low concentration.
(2) Group E shall be exposed to a mixture of test substance in air at the high concentration.
(D) Repeated dosing study. Group F shall receive a series of single daily oral low doses of nonradioactive test substance over a period of at least 7 consecutive days. Twenty four hours after the last nonradioactive dose, a single oral low dose of radioactive test substance shall be administered. Following dosing with radioactive substance, the rats shall be placed in individual metabolic units as described in paragraph (c)(2)(iii) of this section. The study shall be terminated 7 days after the last dose, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first.
(3) Types of studies – (i) Pharmacokinetics studies. Groups A through F shall be used to determine the kinetics of absorption of the test substance. In groups administered the substance by intravenous or oral routes, (i.e., Groups A, B, C, F), the concentration of radioactivity in blood and excreta including expired air shall be measured following administration. In groups administered the substance by the inhalation route (i.e., Groups D and E), the concentration of radioactivity in blood shall be measured at selected time intervals during and following the exposure period. In the groups administered the substance by inhalation (i.e., Groups D and E), the concentration of radioactivity in excreta (including expired air) shall be measured at selected time intervals following the exposure period. In addition, in the groups administered the substance by inhalation, the concentration of test substance in inspired air shall be measured at selected time intervals during the exposure period.
(ii) Metabolism studies. Groups A through F shall be used to determine the metabolism of the test substance. Excreta (urine, feces, and expired air) shall be collected for identification and quantification of test substance and metabolites.
(4) Measurements – (i) Pharmacokinetics. Four animals from each group shall be used for these purposes.
(A) Bioavailability. The levels of radioactivity shall be determined in whole blood, blood plasma or blood serum at 15 minutes, 30 minutes, 1, 2, 3, 6, 9, and 18 hours after dosing; and at 30 minutes, 3, 6, 6.5, 7, 8, 9, 12, and 18 hours after initation of inhalation exposure.
(B) Extent of absorption. The total quantities of radioactivity shall be determined for excreta collected daily for 7 days, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first.
(C) Excretion. The quantities of radioactivity eliminated in the urine, feces, and expired air shall be determined separately at appropriate time intervals. The collection of the intact test substance or its metabolites, including carbon dioxide, may be discontinued when less than 1 percent of the administered dose is found to be exhaled as radioactive carbon dioxide in 24 hours.
(D) Tissue distribution. At the termination of each study, the quantities of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin, spleen, and residual carcass of each animal shall be determined.
(E) Changes in pharmacokinetics. Results of pharmacokinetics measurements (i.e., biotransformation, extent of absorption, tissue distribution, and excretion) obtained in rats receiving the single low oral dose of test substance (Group B) shall be compared to the corresponding results obtained in rats receiving repeated oral doses of test substance (Group F).
(F) Biotransformation. Appropriate qualitative and quantitative methods shall be used to assay urine, feces, and expired air collected from rats. Efforts shall be made to identify any metabolite which comprises 5 percent or more of the dose eliminated.
(G) Changes in biotransformation. Appropriate qualitative and quantitative assay methodology shall be used to compare the composition of radioactive substances in excreta from the rats receiving a single oral dose (Groups B and C) with those in the excreta from rats receiving repeated oral doses (Group F).
(ii) [Reserved]
(d) Data and reporting. The final test report shall include the following:
(1) Presentation of results. Numerical data shall be summarized in tabular form. Pharmacokinetics data shall also be presented in graphical form. Qualitative observations shall also be reported.
(2) Evaluation of results. All quantitative results shall be evaluated by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as specified in the EPA Good Laboratory Practice Standards (40 CFR 792.185), the following specific information shall be reported:
(i) Species and strains of laboratory animals.
(ii) Chemical characterization of the test substance, including:
(A) For the radioactive test substance, information on the site(s) and degree of radiolabeling, including type of label, specific activity, chemical purity, and radiochemical purity.
(B) For the nonradioactive substance, information on chemical purity.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy of all procedures used to generate the data.
(iv) Extent of absorption of the test substance as indicated by: percent absorption of the administered oral dose; and total body burden after inhalation exposure.
(v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood.
(vi) Tissue distribution reported as quantity of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen and in residual carcass of each rat.
(vii) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering single high and low doses to rats.
(viii) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering repeated low doses to rats.
(ix) Pharmacokinetics model(s) developed from the experimental data.
§ 795.232 Inhalation and dermal pharmacokinetics of commercial hexane.
(a) Purposes. The purposes of these studies are to:
(1) Determine the bioavailability of the test substances after dermal and inhalation administration.
(2) Compare the pharmacokinetics and metabolism of the test substances after intravenous, dermal, and inhalation administration.
(3) Examine the effects of repeated doses on the pharmacokinetics and metabolism of the test substances.
(b) Definitions. (1) Bioavailability refers to the relative amount of administered test substance which reaches the systemic circulation and the rate at which this process occurs.
(2) Metabolism means the sum of the enzymatic and nonenzymatic processes by which a particular substance is handled in the body.
(3) Pharmacokinetics means the study of the rates of absorption, tissue distribution, biotransformation, and excretion.
(4) Low dose should correspond to
(5) High dose shall not exceed the lower explosive limit (LEL) and ideally should induce minimal toxicity.
(6) Test substance refers to the unlabeled and both radiolabeled mixtures (
(c) Test procedures – (1) Animal selection – (i) Species. The rat shall be used for pharmacokinetics testing because it has been used extensively for metabolic and toxicological studies.
(ii) Test animals. Adult male and female rats shall be used for testing. The rats shall be 7 to 9 weeks old and their weight range should be comparable from group to group. The animals shall be purchased from a reputable dealer and shall be permanently identified upon arrival. The animals shall be selected at random for the testing groups, and any animal showing signs of ill health shall not be used.
(iii) Animal care. (A) Animal care and housing shall be in accordance with DHHS/PHS NIH Publication No. 86-23, 1985, “Guidelines for the Care and Use of Laboratory Animals.”
(B) The animals shall be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 18 to 26 degrees centigrade and humidity of 40 to 70 percent with a 12-hour light/dark cycle per day. The animal subjects shall be kept in a quarantine facility for at least 7 days prior to use, and shall be acclimated to the experimental environment for a minimum of 48 hours prior to treatment.
(C) During the acclimatization period, the rats shall be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum.
(2) Administration of test substances – (i) Test substances. The study will require he use of both radiolabeled and unlabeled test substances. All unlabeled commercial hexane shall be from the same lot number.Two kinds of radiolabeled test substances will be tested.
(ii) Dosage and treatment – (A) Intravenous. An appropriate dose of the test substance shall be administered intravenously. The intravenous data obtained in this portion of the study shall be suitable for the determination of absorption, distribution, and excretion parameters of the test substance. Factors that should be considered in the selection of the intravenous doses are: The acute toxicity of the test substance, the availability of a suitable vehicle (if saline is unsuitable) and the solubility of the test substance in the vehicle.
(B) Inhalation. Two concentrations of each test substance shall be used in this portion of the study, a low concentration and a high concentration. The high concentration should induce minimal toxicity, but shall not exceed the lower explosive limit (LEL). The low concentration shall correspond to 1/10 of the high concentration. Inhalation treatment shall be conducted using a “nose-cone” or “head only” apparatus to reduce ingestion of the test substance through “grooming” or dermal absorption.
(C) Dermal. Dermal absorption studies should be conducted by the methodology of Susten, A.S., Dames, B.L. and Niemeier, R.W., “In vivo percutaneous absorption studies of volatile solvents in hairless mice. I. Description of a skin depot”, In: Journal of Applied Toxicology 6:43-46, (1986), or by some other suitable method because the test substances have significant volatility. The high and low doses shall be tested in rats.
(iii) Dosing and sampling schedule. Each experimental group shall contain at least four animals of each sex. After administration of the test substance, each rat shall be placed in an individual metabolic unit for collection of urine, feces, and expired air. For the dermal studies, excreta from the rats shall also be collected during the exposure periods. At the end of each collection period, the metabolic units shall be cleaned to recover any excreta that might adhere to the units. All studies, except the repeated dose studies, shall be terminated at 7 days, or after at least 90 percent of the administered radioactivity has been recovered in the excreta, whichever occurs first. All studies described below shall be conducted separately with each radiolabeled test substance.
(A) Intravenous study. Group A shall be given a single intravenous dose of the radiolabeled test substance to result in a level of commercial hexane in the blood that approximates the level from the other routes of exposure so that the data can be used to determine absorption and excretion parameters.
(B) Inhalation studies. A single 6-hour exposure period shall be used for each group.
(1) Group B shall be exposed to a mixture of the radiolabeled test substance in air at the low concentration.
(2) Group C shall be exposed to a mixture of the radiolabeled test substance in air at the high concentration.
(C) Dermal studies. The test substance shall be applied and kept on the skin for a minimum of 6 hours. The covering apparatus components shall be assayed to recover residual radioactivity. At the termination of the studies, each animal shall be sacrificed and the exposed skin area removed. An appropriate section of the skin shall be solubilized and assayed for radioactivity to ascertain whether the skin acts as a reservoir for the test substance.
(1) Group D shall be given one dermal, low dose of the radiolabeled test substance.
(2) Group E shall be given one dermal, high dose of the radiolabeled test substance.
(D) Repeated dosing study. Group F shall receive a series of single daily 6-hour inhalation exposures to unlabeled test substance at the low dose over a period of at least 7 days. A single 6-hour inhalation exposure to the radiolabeled test substance at the low dose shall be administered 24 hours after the last unlabeled exposure. Following administration of the radiolabeled substance, the rats shall be placed in individual metabolic units and excreta collected. The study shall be terminated 7 days after the last exposure, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first.
(3) Types of studies – (i) Pharmacokinetics studies. Groups A through F shall be used to determine the kinetics of absorption of the test substance. In animal subjects administered the test substance intravenously (i.e., Group A), the concentration of test substance in blood and excreta shall be measured following administration. In animal subjects administered the test substance by the inhalation and dermal routes (i.e., Groups B through F), the concentration of test substance in blood shall be measured at selected time intervals during and following the exposure period. In animal subjects administered the test substance by the inhalation route (i.e., Groups B, C, and F) the concentration of test substance in excreta shall be measured following exposure. In animal subjects administered the test substance by the dermal route (i.e., Groups D and E) the concentration of test substance in excreta shall be measured during and following exposure. These measurements allow calculation of uptake, half lives, and clearance. In addition, in the groups administered the test substance by inhalation (i.e., Groups B, C, and F), the concentration of test substance in the exposure chamber air shall be measured at selected time intervals during the exposure period.
(ii) Metabolism studies. Groups A through F shall be used to determine the metabolism of the test substance. Excreta (urine, feces, and expired air) shall be collected for identification and measurement of the quantities of test substance and metabolites.
(4) Measurements – (i) Pharmacokinetics. At least four animals from each group shall be used for these purposes.
(A) Bioavailability. The levels of test substance and relevant metabolites, as appropriate, shall be determined in whole blood, blood plasma or blood serum at appropriate intervals after initiation of intravenous, dermal, and inhalation exposure. The sampling intervals should be compatible with the exposure route under study. The determinations need only be done on animals administered the test substance containing
(B) Extent of absorption. The total quantities of radioactivity shall be determined for excreta collected daily for 7 days, or until at least 90 percent of theradioactivity has been recovered in the excreta, whichever occurs first.
(C) Excretion. The quantities of radioactivity eliminated in the urine, feces, and expired air shall be determined separately at time intervals that provide accurate measurement of clearance and excretory rates. The collection of carbon dioxide may be discontinued when less than one percent of the dose is found to be exhaled as radioactive carbon dioxide in 24 hours.
(D) Tissue distribution. At the termination of each study, the quantities of radioactivity shall be determined in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin, spleen, thymus, and residual carcass of each animal.
(E) Change in pharmacokinetics. Results of pharmacokinetics measurements (i.e., biotransformation, extent of absorption, tissue distribution, and excretion) obtained in rats receiving the single inhalation exposure to the low dose of the test substance (Group B) shall be compared to the corresponding results obtained in rats receiving repeated inhalation exposures to the low dose of the test substance (Group F).
(ii) Metabolism. At least four animals from each group shall be used for these purposes.
(A) Biotransformation. Appropriate qualitative and quantitative methods shall be used to assay urine, feces, and expired air collected from rats. Efforts shall be made to identify any metabolite which comprises 5 percent or more of the dose administered.
(B) Changes in biotransformation. Appropriate qualitative and quantitative assay methods shall be used to compare the composition of radioactive compounds in excreta from rats receiving a single inhalation exposure (Groups B and C) with that from rats receiving repeated inhalation exposures (Group F).
(d) Data and reporting. The final test report shall include the following:
(1) Presentation of results. Numerical data shall be summarized in tabular form. Pharmacokinetics data shall also be presented in graphical form. Qualitative observations shall also be reported.
(2) Evaluation of results. All data shall be evaluated by appropriate statistical methods.
(3) Reporting results. In addition to the reporting requirements as specified in 40 CFR part 792, the following information shall be reported.
(i) Strain of laboratory animals.
(ii) Chemical characterization of the test substances, including:
(A) For the radiolabeled test substances, information on the sites and degree of radiolabeling, including type of label, specific activity, chemical purity prior to mixing with the unlabeled hexane mixture, and radiochemical purity.
(B) For the unlabeled test substance, information on lot number and the percentage of MCP and n-hexane.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy of all procedures used to obtain the data.
(iv) Percent and rate of absorption of the test substance after inhalation and dermal exposures.
(v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. For dermal studies, include recovery data for skin and residual radioactivity in the covering apparatus.
(vi) Tissue distribution reported as quantity of radioactivity in blood, in various tissues including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen, thymus, and in residual carcass.
(vii) Biotransformation pathways, to the extent possible, and quantities of the test substances and metabolites in excreta collected after administering single high and low doses.
(viii) Biotransformation pathways, to the extent possible, and quantities of test substances and metabolites in excreta collected after administering repeated low doses.
(ix) Pharmacokinetics models to the extent they can be developed from the experimental data.
§ 795.250 Developmental neurotoxicity screen.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, it is important to determine when acceptable exposures in the adult may not be acceptable to a developing organism. This test is designed to provide information on the potential functional and morphologic hazards to the nervous system which may arise in the offspring from exposure of the mother during pregnancy and lactation.
(b) Principle of the test method. The test substance is administered to several groups of pregnant animals during gestation and lactation, one dose level being used per group. Offspring are randomly selected from within litters for neurotoxicity evaluation. The evaluation includes observation to detect gross neurological and behavioral abnormalities, determination of motor activity, neuropathological evaluation, and brain weights. Measurements are carried out periodically during both postnatal development and adulthood.
(c) Test procedures – (1) Animal selection – (i) Species and strain. Testing should be performed in the Sprague Dawley rat.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant females shall be used at each dose level.
(iv) Number of animals. The objective is for a sufficient number of pregnant rats to be exposed to ensure that an adequate number of offspring are produced for neurotoxicity evaluation. At least 20 litters are recommended at each dose level. This number assumes a coefficient of variation of 20 to 25 percent for most behavioral tests. If, based upon experience with historical control data or data for positive controls in a given laboratory, the coefficient of variation for a given task is higher than 20 to 25 percent, then calculation of appropriate sample sizes to detect a 20 percent change from control values with 80 percent power would need to be done. For most designs, calculations can be made according to Dixon and Massey (1957) under paragraph (e)(5) of this section, Neter and Wasserman (1974) under paragraph (e)(10) of this section, Sokal and Rohlf (1969) under paragraph (e)(11) of this section, or Jensen (1972) under paragraph (e)(8) of this section.
(A) On day 4 after birth, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Whenever the number of male or female pups prevents having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) is permitted. Adjustments are not appropriate for litters of less than 8 pups. Elimination of runts only is not appropriate. Individual pups should be identified uniquely after standardization of litters. A method that may be used can be found in Adams et al. (1985) under paragraph (e)(1) of this section.
(B) After standardization of litters, males and females shall be randomly assigned to one of each of three behavioral tasks. Alternatively, more than one of the behavioral tasks may be conducted in the same animal. In the latter case, a minimum of 1 to 2 days should separate the tests when conducted at about the same age.
(C) One male and one female shall be randomly selected from each litter for sacrifice at weaning as specified in paragraph (c)(8) of this section.
(2) Control group. A concurrent control group shall be used. This group shall be a sham treated group, or, if a vehicle is used in administering the test substance, a vehicle control group. Animals in the control groups shall be handled in an identical manner to test group animals. The vehicle shall neither be developmentally toxic nor have effects on reproduction.
(3) Dose levels and dose selection. (i) At least 3 dose levels plus a control (vehicle control, if a vehicle is used) shall be used.
(ii) If the substance has been shown to be developmentally toxic either in a standard developmental toxicity study or a pilot study, the highest dose level shall be the maximum dose which will not induce in utero or neonatal deaths or malformations sufficient to preclude a meaningful evaluation of neurotoxicity.
(iii) In the absence of standard developmental toxicity, unless limited by the physicochemical nature or biologicial properties of the substance, the highest dose level shall induce some overt maternal toxicity but shall not result in a reduction in weight gain exceeding 20 percent during gestation and lactation.
(iv) The lowest dose should not produce any grossly observable evidence of either maternal or developmental neurotoxicity.
(v) The intermediate dose(s) shall be equally spaced between the highest and lowest dose.
(4) Dosing period. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The dose period shall cover the period from day 6 of gestation through weaning (21 days postnatally).
(5) Administration of test substance. The test substance or vehicle should be administered orally by intubation. The test substance shall be administered at the same time each day. The animals shall be weighed periodically and the dosage based on the most recent weight determination.
(6) Observation of dams. (i) A gross examination of the dams shall be made at least once each day, before daily treatment. The animals shall be observed by trained technicians who are blind with respect to the animal’s treatment, using standardized procedures to maximize inter-observer reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of inter-observer reliability is required.
(ii) During the treatment and observation periods, cage-side observations shall include:
(A) Any responses with respect to body position, activity level, coordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to headflicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards.
(C) The presence of:
(1) Convulsions.
(2) Tremors.
(3) Increased levels of lacrimation and/or red-colored tears.
(4) Increased levels of salivation.
(5) Piloerection.
(6) Pupillary dilation or constriction.
(7) Unusual respiration (shallow, labored, dyspneic, gasping, and retching) and/or mouth breathing.
(8) Diarrhea.
(9) Excessive or diminished urination.
(10) Vocalization.
(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree and duration.
(iv) Animals shall be weighed at least weekly.
(v) The day of delivery of litters shall be recorded.
(7) Study conduct – (i) Observation of offspring. (A) All offspring shall be examined cage-side daily for gross signs of mortality and morbidity.
(B) All offspring shall be examined outside the cage for gross signs of toxicity whenever they are weighed or removed from their cages for behavioral testing. The offspring shall be observed by trained technicians, who are blind with respect to the animal’s treatment using standardized procedures to maximize inter-observer reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of inter-observer reliability is required. At a minimum, the end points outlined in paragraph (c)(6)(ii) of this section shall be monitored as appropriate for the developmental stage being observed.
(C) Any gross signs of toxicity in the offspring shall be recorded as they are observed, including the time of onset, the degree, and duration.
(ii) Developmental landmarks. Live pups should be counted and litters weighed by weighing each individual pup at birth, or soon thereafter, and on days 4, 7, 13, 17, and 21, and biweekly thereafter. The age of the pups at the time of the appearance of the following developmental landmarks shall be determined:
(A) Vaginal opening. General procedure for this determination may be found in Adams et al. (1985) under paragraph (e)(1) of this section.
(B) Testes descent. General procedure for this determination may be found in Adams et al. (1985) under paragraph (e)(1) of this section.
(iii) Motor activity. (A) Motor activity shall be monitored specifically on days 13, 17, 21, 45 (±2 days), and 60 (±2 days). Motor activity shall be monitored by an automated activity recording apparatus. The device used shall be capable of detecting both increases and decreases in activity, i.e., baseline activity as measured by the device shall not be so low as to preclude decreases nor so high as to preclude increases. Each device shall be tested by standard procedures to ensure, to the extent possible, reliability of operation across devices and testing of animals within dose groups shall be balanced across devices.
(B) Each animal shall be tested individually. The test session shall be long enough to demonstrate habituation of motor activity in control animals, i.e., to approach asymptotic levels by the last 20 percent of the session. Animals’ activity counts shall be collected in equal time periods of no greater than 10 minutes duration. All sessions shall have the same duration. Treatment groups shall be counter-balanced across test times.
(C) Efforts shall be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables which can affect motor activity are sound level, size, and shape of the test cage, temperature, relative humidity, lighting conditions, odors, use of home cage or novel test cage, and environmental distractions.
(D) Additional information on the conduct of a motor activity study may be obtained in the TSCA motor activity guideline, in § 798.6200 of this chapter.
(iv) Auditory startle test. An auditory startle habituation test shall be performed on the offspring on days 22 and 60. Details on the conduct of this testing may be obtained in Adams et al. (1985) under paragraph (e)(1) of this section. In performing the auditory startle task, the mean response amplitude on each block of 10 trials (5 blocks of 10 trials per session on each day of testing) shall be made. While use of pre-pulse inhibition is not a requirement, it may be used at the discretion of the investigator. Details on the conduct of this testing may be obtained from Ison (1984) under paragraph (e)(7) of this section.
(v) Active avoidance test. Active avoidance testing shall be conducted beginning at 60 to 61 days of age. Details on the apparatus may be obtained in Brush and Knaff (1959) and on the conduct of testing from Brush (1962), under paragraphs (e)(2) and (e)(4) of this section, respectively; reviews on active avoidance conditioning by Brush (1971) and McAllister and McAllister (1971) can be found under paragraphs (e)(3) and (e)(9) of this section, respectively. In performing the active avoidance task, the following measures should be made:
(A) Mean number of shuttles during the adaptation period preceding each daily session.
(B) Mean number and latency of avoidances per session, presented in blocks of 10 trials (2 blocks of 10 trials per session across 5 sessions).
(C) Mean number and latency of escapes per session, presented in blocks of 10 trials as above.
(D) Mean duration of shocks per session, presented in blocks of 10 trials as above.
(E) Mean number of shuttles during the inter-trial intervals.
(8) Post-mortem evaluation – (i) Age of animals. One male and one female per litter shall be sacrificed at weaning and the remainder following the last behavioral measures. Neuropathology and brain weight determinations shall be made on animals sacrificed at weaning and after the last behavioral measures.
(ii) Neuropathology. Details for the conduct of neuropathology evaluation may be obtained in the TSCA neuropathology guideline, in § 798.6400 of this chapter. At least 6 offspring per dose group shall be randomly selected from each sacrificed group (weaning and adulthood) for neuropathologic evaluation. These animals shall be balanced across litters, and equal numbers of males and females shall be used. The remaining sacrificed animals shall be used to determine brain weight. Animals shall be perfused in situ by a generally recognized technique. After perfusion, the brain and spinal cord shall be removed and gross abnormalities noted. Cross-sections of the following areas shall be examined: The forebrain, the center of the cerebrum and midbrain, the cerebellum and pons, and the medulla oblongata; the spinal cord at cervical and lumbar swelling; Gasserian ganglia, dorsal root ganglia, dorsal and ventral root fibers, proximal sciatic nerve (mid-thigh and sciatic notch), sural nerve (at knee), and tibial nerve (at knee). Tissue samples from both the central and peripheral nervous system shall be further immersion-fixed and stored in appropriate fixative for further examination. After dehydration, tissue specimens shall be cleared with xylene and embedded in paraffin or paraplast except for the sural nerve which should be embedded in plastic. A method for plastic embedding is described by Spencer et al. under paragraph (e)(12) of this section. Tissue sections shall be prepared from the tissue blocks. The following general testing sequence is recommended for gathering histopathological data:
(A) General staining. A general staining procedure shall be performed on all tissue specimens in the highest treatment group. Hematoxylin and eosin (H&E) shall be used for this purpose. The staining shall be differentiated properly to achieve bluish nuclei with pinkish background.
(B) Special stains. Based on the results of the general staining, selected sites and cellular components shall be further evaluated by use of specific techniques. If H&E screening does not provide such information, a battery of stains shall be used to assess the following components in all appropriate required samples: Neuronal body (e.g., Einarson’s gallocyanin), axon (e.g., Kluver’s Luxol Fast Blue), and neurofibrils (e.g., Bielchosky). In addition, nerve fiber teasing shall be used. A section of normal tissue shall be included in each staining to assure that adequate staining has occurred. Any changes shall be noted and representative photographs shall be taken. If lesions are observed, the special techniques shall be repeated in the next lower treatment group until no further lesions are detectable.
(C) Alternative technique. If the anatomical locus of expected neuropathology is well-defined, epoxy-embedded sections stained with toluidine blue may be used for small sized tissue samples. This technique obviates the need for special stains.
(iii) Brain weight. At least 10 animals that are not sacrificed for histopathology shall be used to determine brain weight. The animals shall be decapitated and the brains carefully removed, blotted, chilled, and weighed. The following dissection shall be performed on an ice-cooled glass plate: First, the rhombencephalon is separated by a transverse section from the rest of the brain and dissected into the cerebellum and the medulla oblongata/pons. A transverse section is made at the level of the “optic chiasma” which delimits the anterior part of the hypothalamus and passes through the anterior commissure. The cortex is peeled from the posterior section and added to the anterior section. This divides the brain into four sections, the telencephalon, the diencephalon/mid-brain, the medulla oblongata/pons, and the cerebellum. Sections shall be weighed as soon as possible after dissection to avoid drying. Detailed methodology is available in Glowinski and Iversen (1966) under paragraph (e)(6) of this section.
(d) Data reporting and evaluation. In addition to the reporting requirements specified in part 792, subpart J of this chapter, the final test report shall include the following information.
(1) Description of system and test methods. (i) A detailed description of the procedures used to standardize observation and operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. These data do not have to be from studies using prenatal exposures. However, the laboratory must demonstrate competence in testing neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group.
(iii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups.
(iv) A short justification explaining any decisions where professional judgement is involved such as fixation technique and choice of stains.
(2) Results. The following information shall be arranged by test group dose level.
(i) In tabular form, data for each animal shall be provided showing:
(A) Its identification number and litter from which it came.
(B) Its body weight and score on each developmental landmark at each observation time; total session activity counts and intrasession subtotals on each day measured; auditory startle response magnitude session counts and intrasession subtotals on each day measured; avoidance session counts and intrasession counts on each day measured; time and cause of death (if appropriate); locations, nature or frequency, and severity of the lesions; total brain weight; absolute weight of each of the four sections; and weight of each section as a percentage of total brain weight. A commonly used scale such as 1 + , 2 + , 3 + , and 4 + for degree of severity of lesions ranging from very slight to extensive may be used for morphologic evaluation. Any diagnoses derived from neurologic signs and lesions, including naturally occurring diseases or conditions, shall also be recorded.
(ii) Summary data for each group shall include:
(A) The number of animals at the start of the test.
(B) Body weights of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each observation score at each observation time.
(E) The percentage of animals showing each abnormal sign at each observation time.
(F) The mean and standard deviation for each continuous end point at each observation time. These will include body weight, motor activity counts, acoustic startle responses, performance in active avoidance tests, and brain weights (both absolute and relative).
(G) The number of animals in which any lesion was found.
(H) The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesions.
(3) Evaluation of data. An evaluation of the test results shall be made. The evaluation shall include the relationship between the doses of the test substance and the presence or absence, incidence, and severity of any neurotoxic effect. The evaluation shall include appropriate statistical analyses. The choice of analyses shall consider tests appropriate to the experimental design and needed adjustments for multiple comparisons.
(e) References. For additional background information on this test guideline, the following references should be consulted:
(1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. “Collaborative behavioral teratology study: Protocol design and testing procedure.” Neurobehavioral Toxicology and Teratology. 7: 579-586. (1985).
(2) Brush, F.R. “The effects of inter-trial interval on avoidance learning in the rat.” Journal of Comparative Physiology and Psychology. 55: 888-892. (1962).
(3) Brush, F.R. “Retention of aversively motivated behavior.” In: “Adverse Conditioning and Learning.” Brush, F.R., ed., New York: Academic Press. (1971).
(4) Brush, F.R. and Knaff, P.R. “A device for detecting and controlling automatic programming of avoidance-conditioning in a shuttle-box.” American Journal of Psychology. 72: 275-278 (1959).
(5) Dixon, W.J. and Massey, E.J. “Introduction to Statistical Analysis.” 2nd ed. New York: McGraw-Hill. (1957).
(6) Glowinski, J. and Iversen, L.L. “Regional studies of catecholamines in the rat brain-I.” Journal of Neurochemistry. 13: 655-669. (1966).
(7) Ison, J.R. “Reflex modification as an objective test for sensory processing following toxicant exposure.” Neurobehavioral Toxicology and Teratology. 6: 437-445. (1984).
(8) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling’s T2 Statistics.” Biometrics. 28: 39-53. (1972).
(9) McAllister, W.R. and McAllister, D.E. “Behavioral measurement of conditioned fear.” In: “Adverse Conditioning and Learning.” Brush, F.R., ed., New York: Academic Press (1971).
(10) Neter, J. and Wasserman, W. “Applied Linear Statistical Models.” Homewood: Richard D. Irwin, Inc. (1974).
(11) Sokal, R.P. and Rohlf, E.J. “Biometry.” San Francisco: W.H. Freeman and Co. (1969).
(12) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H., “Neuropathological methods for the detection of neurotoxic disease.” In: “Experimental and Clinical Neurotoxicology.” Spencer, P.S. and Schaumburg, H.H., eds., Baltimore, MD: Williams & Wilkins, pp. 743-757. (1980).
PART 796 – CHEMICAL FATE TESTING GUIDELINES
Subpart A [Reserved]
Subpart B – Physical and Chemical Properties
§ 796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra.
(a) Introductory information – (1) Guidance information. (i) Molecular formula.
(ii) Structural formula.
(2) Standard documents. The spectrophotometric method is based on national standards and consensus methods which are applied to measure the absorption spectra.
(b) Method – (1)(i) Introduction, purpose, scope, relevance, application and limits of test. (A) The primary environmental purpose in determining the ultraviolet-visible (UV-VIS) absorption spectrum of a chemical compound is to have some indication of the wavelengths at which the compounds may be susceptible to photochemical degradation. Since photochemical degradation is likely to occur in both the atmosphere and the aquatic environment, spectra appropriate to these media will be informative concerning the need for further persistence testing.
(B) Degradation will depend upon the total energy absorbed in specific wavelength regions. Such energy absorption is characterized by both molar absorption coefficient (molar extinction coefficient) and band width. However, the absence of measurable absorption does not preclude the possibility of photodegradation.
(ii) Definitions and units. The UV-VIS absorption spectrum of a solution is a function of the concentration, c
(iii) Reference substances. (A) The reference substances need not be employed in all cases when investigating a new substance. They are provided primarily so that calibration of the method may be performed from time to time and to offer the chance to compare the results when another method is applied.
(B) Reference compounds appropriate for the calibration of the system are:
(1) Potassium dichromate (in 0.005 mol/L, H
| log ε | 3.56 | 3.63 | 3.16 | 3.50 |
| λ in nm | 235 | 257 | 313 | 350 |
(2) Fluoranthene (in methanol) from C.R.C. Atlas of Spectral Data, paragraph (d)(3) of this section:
| log ε | 4.75 | 4.18 | 4.73 | 3.91 | 3.92 |
| λ in nm | 237 | 236 | 288 | 339 | 357 |
(3) 4-nitrophenol (in methanol) from C.R.C. Atlas of Spectral Data, paragraph (d)(3) of this section:
| log ε | 3.88 | 4.04 |
| λ in nm | 288 | 311 |
(iv) Principle of the test method. This method utilizes a double-beam spectrophotometer which records only the absorption differences between the blank and test solutions to give the spectrum of the chemical being tested.
(v) Quality criteria – Reproducibility and sensitivity. (A) Reproducibility and sensitivity, need not be measured directly. Instead, the accuracy of the system in measuring the spectra of reference compounds will be defined so as to assure appropriate reproducibility and sensitivity. It is preferable to use a recording double-beam spectrophotometer to obtain the UV-VIS spectrum of the test compound. Such an instrument should have a photometric accuracy of ±0.02 units over the absorbance range of 0 to 2 units. It should be capable of recording absorbances at wavelengths of 200 to 750 nanometers nm with a wavelength accuracy of ±0.5 nm. The cells employed with the instrument must necessarily be transparent over this wavelength range and must have a path length determined to within 1 percent. To ensure that the instrument is performing satisfactorily, spectra for test solutions of K
(B) In the event that a recording double-beam instrument is not available, it will be necessary to determine the absorbance of the test solution in a single-beam instrument at 5-nm intervals over the entire wavelength range and at 1-nm intervals where there are indicated absorbance maxima. Wavelength and absorbance tests should be done as with the double-beam instrument.
(2) Description of the test procedure – (i) Preparation – (A) Preparation of test solutions. (1) Solutions should be prepared by accurately weighing an appropriate amount of the purest form of the test substance available. This should be made up in a concentration which will result in at least one absorbance maximum in the range 0.5 to 1.5 units.
(2) The absorption of a compound is due to its particular chemical form. It is often the case that different forms are present, depending on whether the medium is acidic, basic, or neutral. Consequently, spectra under all three conditions are required where solubility and concentration allow. Where it is not possible to obtain sufficient concentrations in any of the aqueous media, a suitable organic solvent should be used (methanol preferred).
(3) The acid medium should have a pH of less than 2, and the basic medium should be at least pH 10. The solvent for the neutral solution, and for preparing the acidic and basic ones, should be distilled water, transparent to ultraviolet radiation down to 200 nm. If methanol must be used, acidic and basic solutions can be prepared by adding 10 percent by volume of HCl or NaOH in aqueous solution ([HCl], [NaOH] = 1 mol/L).
(4) In theory, all chemical species other than that being tested are present in both beams and would therefore not appear in the recorded spectrum of a double-beam instrument. In practice, because the solvent is usually present in great excess, there is a threshold value of wavelength below which it is not possible to record the spectrum of the test chemical. Such a wavelength will be a property of the solvent or of the test medium. In general, distilled water is useful from 200 nm (dissolved ions will often increase this), methanol from 210 nm, hexane from 210 nm, acetonitrile from 215 nm and dichloromethane from 235 nm.
(B) Blank solutions. A blank must be prepared which contains the solvent and all chemical species other than the test chemical. The absorption spectrum of this solution should be recorded in a manner identical to that of the test solution and preferably on the same chart. This “baseline” spectrum should never record an absorbance reading varying more than ±0.05 from the nominal zero value.
(C) Cells. Cell pathlengths are usually between 0.1 cm and 10 cm. Cell lengths should be selected to permit recording of at least one maximum in the absorbance range of 0.5 to 1.5 units. Which set of cells should be used will be governed by the concentration and the absorbance of the test solution as indicated by the Beer-Lambert Law. The cells should be transparent over the range of the spectrum being recorded, and the path-lengths should be known to an accuracy of at least 1 per cent. Cells should be thoroughly cleaned in an appropriate manner (chromic acid is useful for quartz cells) and rinsed several times with the test or blank solutions.
(ii) Performance of the test. Both cells to be employed should be rinsed with the blank solution and then filled with same. The instrument should be set to scan at a rate appropriate for the required wavelength resolution and the spectrum of the blank recorded. The sample cell should then be rinsed and filled with the test solution and the scanning repeated, preferably on the same spectrum chart, to display the baseline. The test should be carried out at 25 °C.
(c) Data and reporting – (1) Treatment of results. (i) The molar absorption coefficient ε should be calculated for all absorbance maxima of the test substance. The formula for this calculation is:
(ii) For each peak which is capable of being resolved, either as recorded or by extrapolated symmetrical peaks, the bandwidth should be recorded.
(2) Test report. (i) The report should contain a copy of each of the three spectra (3 pH conditions). If neither water nor methanol solutions are feasible, there will be only one spectrum. Spectra should include a readable wave-length scale. Each spectrum should be clearly marked with the test conditions.
(ii) For each maximum in each spectrum, the ε value and bandwidth (when applicable) should be calculated and reported, along with the wavelength of the maximum. This should be presented in tabular form.
(iii) The various test conditions should be included, such as scan speed, the name and model of the spectrophotom-eter, the slit width (where available), cell type and path length, the concentrations of the test substance, and the nature and acidity of the solvent medium. A recent test spectrum on appropriate reference materials for photometric and wavelength accuracy should also be submitted (see Reproducibility and sensitivity).
(d) Literature references. For additional background information on this test guideline, the following references should be consulted:
(1) Milazzo, G., Caroli, S., Palumbo-Doretti, M., Violante, N., Analytical Chemistry, 49: 711 (1977).
(2) Katelaar, J.A.A., Photoelectric Spectrometry Group Bulletin, 8, (Cambridge, 1955).
(3) Chemical Rubber Company, Atlas of Spectral Data, (Cliffland, Ohio).
§ 796.1950 Vapor pressure.
(a) Introduction – (1) Background and purpose. (i) Volatilization, the evaporative loss of a chemical, depends upon the vapor pressure of chemical and on environmental conditions which influence diffusion from a surface. Volatilization is an important source of material for airborne transport and may lead to the distribution of a chemical over wide areas and into bodies of water far from the site of release. Vapor pressure values provide indications of the tendency of pure substances to vaporize in an unperturbed situation, and thus provide a method for ranking the relative volatilities of chemicals. Vapor pressure data combined with water solubility data permit the calculation of Henry’s law constant, a parameter essential to the calculation of volatility from water.
(ii) Chemicals with relatively low vapor pressures, high adsorptivity onto solids, or high solubility in water are less likely to vaporize and become airborne than chemicals with high vapor pressures or with low water solubility or low adsorptivity to solids and sediments. In addition, chemicals that are likely to be gases at ambient temperatures and which have low water solubility and low adsorptive tendencies are less likely to transport and persist in soils and water. Such chemicals are less likely to biodegrade or hydrolyze and are prime candidates for atmospheric oxidation and photolysis (e.g., smog formation or stratospheric alterations). On the other hand, nonvolatile chemicals are less frequently involved in atmosphere transport, so that concerns regarding them should focus on soils and water.
(iii) Vapor pressure data are an important consideration in the design of other chemical fate and effects tests; for example, in preventing or accounting for the loss of violatile chemicals during the course of the test.
(2) Definitions and units. (i) “Desorption efficiency” of a particular compound applied to a sorbent and subsequently extracted with a solvent is the weight of the compound which can be recovered from the sorbent divided by the weight of the compound originally sorbed.
(ii) “Pascal” (Pa) is the standard international unit of vapor pressure and is defined as newtons per square meter (N/m
(iii) The “torr” is a unit of pressure which equals 133.3 pascals or 1 mm Hg at 0 °C.
(iv) “Vapor pressure” is the pressure at which a liquid or solid is in equilibrium with its vapor at a given temperature.
(v) “Volatilization” is the loss of a substance to the air from a surface or from solution by evaporation.
(3) Principle of the test methods. (i) The isoteniscope procedure uses a standardized technique [ASTM 1978] that was developed to measure the vapor pressure of certain liquid hydrocarbons. The sample is purified within the equipment by removing dissolved and entrained gases until the measured vapor pressure is constant, a process called “degassing.” Impurities more volatile than the sample will tend to increase the observed vapor pressure and thus must be minimized or removed. Results are subject to only slight error for samples containing nonvolatile impurities.
(ii) Gas saturation (or transpiration) procedures use a current of inert gas passed through or over the test material slowly enough to ensure saturation and subsequent analysis of either the loss of material or the amount (and sometimes kind) of vapor generated. Gas saturation procedures have been described by Spencer and Cliath (1969) under paragraph (d)(2) of this section. Results are easy to obtain and can be quite precise. The same procedures also can be used to study volatilization from laboratory scale environmental simulations. Vapor pressure is computed on the assumption that the total pressure of a mixture of gases is equal to the sum of the pressures of the separate or component gases and that the ideal gas law is obeyed. The partial pressure of the vapor under study can be calculated from the total gas volume and the weight of the material vaporized. If v is the volume which contains w grams of the vaporized material having a molecular weight M, and if p is the pressure of the vapor in equilibrium at temperature T (K), then the vapor pressure, p, of the sample is calculated by
(iii) In an effort to improve upon the procedure described by Spencer and Cliath (1969) under paragraph (d)(2) of this section, and to determine the applicability of the gas saturation method to a wide variety of chemical types and structures, EPA has sponsored research and development work at SRI International (EPA 1982) under paragraph (d)(1) of this section. The procedures described in this Test Guideline are those developed under that contract and have been evaluated with a wide variety of chemicals of differing structure and vapor pressures.
(4) Applicability and specificity. (i) A procedure for measuring the vapor pressure of materials released to the environment ideally would cover a wide range of vapor pressure values, at ambient temperatures. No single procedure can cover this range, so two different procedures are described in this section, each suited for a different part of the range. The isoteniscope procedure is for pure liquids with vapor pressures from 0.1 to 100 kPa. For vapor pressures of 10
(ii) With respect to the isoteniscope method, if compounds that boil close to or form azeotropes with the test material are present, it is necessary to remove the interfering compounds and use pure test material. Impurities more volatile than the sample will tend to increase the observed vapor pressure above its true value but the purification steps will tend to remove these impurities. Soluble, nonvolatile impurities will decrease the apparent vapor pressure. However, because the isoteniscope procedure is a static, fixed-volume method in which an insignificant fraction of the liquid sample is vaporized, it is subject to only slight error for samples containing nonvolatile impurities. That is, the nonvolatile impurities will not be concentrated due to vaporization of the sample.
(iii) The gas saturation method is applicable to solid or liquid chemicals. Since the vapor pressure measurements are made at ambient temperatures, the need to extrapolate data from high temperatures is not necessary and high temperature extrapolation, which can often cause serious errors, is avoided. The method is most reliable for vapor pressures below 10
(b) Test procedures – (1) Test conditions. (i) The apparatus in the isoteniscope method is described in paragraph (b)(2)(i) of this section.
(ii) The apparatus used in the gas saturation method is described in paragraph (b)(2)(ii) of this section.
(2) Performance of the tests – (i) Isoteniscope Procedure. The isoteniscope procedure described as ANSI/ASTM Method D 2879-86 is applicable for the measurement of vapor pressures of liquids with vapor pressures of 0.1 to 100 kilopascals (kPa) (0.75 to 750 torr). ASTM D 2879-86 is available for inspection at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. This incorporation by reference was approved by the Director of the Office of the Federal Register. This material is incorporated as it exists on the date of approval and a notice of any change in this material will be published in the
(ii) Gas saturation procedure. (A) The test procedures require the use of a constant-temperature box as depicted in the following Figure 1.
(B) Solid samples are loaded into 5 mm i.d. glass tubing between glass wool plugs. The following Figure 2 depicts a drawing of a sample holder and absorber system.
(C) Liquid samples are contained in a holder as shown in the following Figure 3.
(D) At very low vapor pressures and sorbent loadings, adsorption of the chemical on the glass wool separating the sample and the sorbent and on the glass surfaces may be a serious problem. Therefore, very low loadings should be avoided whenever possible. Incoming nitrogen gas (containing no interfering impurities) passes through a coarse frit and bubbles through a 38 cm column of liquid sample. The stream passes through a glass wool column to trap aerosols and then through a sorbent tube, as described above. The pressure drop across the glass wool column and the sorbent tube are negligible.
(E) With both solid and liquid samples, at the end of the sampling time, the front and backup sorbent sections are analyzed separately. The compound on each section is desorbed by adding the sorbent from that section to 1.0 ml of desorption solvent in a small vial and allowing the mixture to stand at a suitable temperature until no more test compound desorbs. It is extremely important that the desorption solvent contain no impurities which would interfere with the analytical method of choice. The resulting solutions are analyzed quantitatively by a suitable analytical method to determine the weight of sample desorbed from each section. The choice of the analytical method, sorbent, and desorption solvent is dictated by the nature of the test material. Commonly used sorbents include charcoal, Tenax GC, and XAD-2. Describe in detail the sorbent, desorption solvent, and analytical methods employed.
(F) Measure the desorption efficiency for every combination of sample, sorbent, and solvent used. The desorption efficiency is determined by injecting a known mass of sample onto a sorbent and later desorbing it and analyzing for the mass recovered. For each combination of sample, sorbent, and solvent used, carry out the determination in triplicate at each of three concentrations. Desorption efficiency may vary with the concentration of the actual sample and it is important to measure the efficiency at or near the concentration of sample under gas saturation test procedure conditions.
(G) To assure that the gas is indeed saturated with test compound vapor, sample each compound at three differing gas flow rates. Appropriate flow rates will depend on the test compound and test temperature. If the calculated vapor pressure shows no dependence on flow rate, then the gas is assumed to be saturated.
(c) Data and reporting. (1) Report the triplicate calculated vapor pressures for the test material at each temperature, the average calculated vapor pressure at each temperature, and the standard deviation.
(2) Provide a description of analytical methods used to analyze for the test material and all analytical results.
(3) For the isoteniscope procedure, include the plot of p vs. the reciprocal of the temperature in K, developed during the degasing step and showing linearity in the region of 298.15 K (25 °C) and any other required test temperatures.
(4) For the gas saturation procedure, include the data on the calculation of vapor pressure at three or more gas flow rates at each test temperature, showing no dependence on flow rate. Include a description of sorbents and solvents employed and the desorption efficiency calculations.
(5) Provide a description of any difficulties experienced or any other pertinent information.
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency. Evaluation of Gas Saturation Methods to Measure Vapor Pressures: Final Report, EPA Contract No. 68-01-5117 with SRI International, Menlo Park, California (1982).
(2) Spencer, W.F. and Cliath, M.M. “Vapor Density of Dieldrin,” Journal of Agricultural and Food Chemistry, 3:664-670 (1969).
(3) Spencer, W.F. and Cliath, M.M. “Vapor Density and Apparent Vapor Pressure of Lindane,” Journal of Agricultural and Food Chemistry, 18:529-530 (1970).
Subpart C – Transport Processes
§ 796.2750 Sediment and soil adsorption isotherm.
(a) Introduction – (1) Background and purpose. The adsorption of chemicals to sediments and soils is an important process that affects a chemical’s distribution in the environment. If a chemical is adsorbed to soil particles, it will remain on the soil surface and will not reach ground water. If a chemical is not adsorbed, it will leach through the soil profile and may reach ground waters and then surface waters. Similarly, if a chemical adsorbed to sediment, it will accumulate in the bed and suspended load of aquatic systems. If a chemical is not adsorbed to sediment, it will accumulate in the water column of aquatic systems. Information on the adsorption potential is needed under certain circumstances to assess the transport of chemicals in the environment. This section describes procedures that will enable sponsors to determine the adsorption isotherm of a chemical on sediments and soils.
(2) Definitions and units. (i) The “cation exchange capacity” (CEC) is the sum total of exchangeable cations that a sediment or soil can adsorb. The CEC is expressed in milliequivalents of negative charge per 100 grams (meq/100g) or milliequivalents of negative charge per gram (meq/g) of soil or sediment.
(ii) “Clay mineral analysis” is the estimation or determination of the kinds of clay-size minerals and the amount present in a sediment or soil.
(iii) “Organic matter” is the organic fraction of the sediment or soil; it includes plant and animal residues at various stages of decomposition, cells and tissues of soil organisms, and substances synthesized by the microbial population.
(iv) “Particle size analysis” is the determination of the various amounts of the different particle sizes in a sample (i.e., sand, silt, clay), usually by sedimentation, sieving, micrometry, or combinations of these methods. The names and diameter range commonly used in the United States are:
| Name | Diameter range |
|---|---|
| Very coarse sand | 2.0 to 1.0 mm |
| Coarse sand | 1.0 to 0.5 mm |
| Medium sand | 0.5 to 0.25 mm |
| Fine sand | 0.25 to 0.125 mm |
| Very fine sand | 0.125 to 0.062 mm |
| Silt | 0.062 to 0.002 mm |
| Clay |
(v) The “pH” of a sediment or soil is the negative logarithm to the base ten of the hydrogen ion activity of the sediment or soil suspension. It is usually measured by a suitable sensing electrode coupled with a suitable reference electrode at a 1/1 solid/solution ratio by weight.
(vi) The adsorption ratio, “K
(vii) “Sediment” is the unconsolidated inorganic and organic material that is suspended in and being transported by surface water, or has settled out and has deposited into beds.
(viii) “Soil” is the unconsolidated mineral material on the immediate surface of the earth that serves as a natural medium for the growth of land plants. Its formation and properties are determined by various factors such as parent material, climate, macro- and microorganisms, topography, and time.
(ix) “Soil aggregate” is the combination or arrangement of soil separates (sand, silt, clay) into secondary units. These units may be arranged in the soil profile in a distinctive characteristic pattern that can be classified according to size, shape, and degree of distinctness into classes, types, and grades.
(x) “Soil classification” is the systematic arrangement of soils into groups or categories. Broad groupings are based on general soil characteristics while subdivisions are based on more detailed differences in specific properties. The soil classification system used in this standard and the one used today in the United States is the 7th Approximation-Comprehensive System. The ranking of subdivisions under this system is: Order, Suborder, Great group, family, and series.
(xi) A “soil horizon” is a layer of soil approximately parallel to the land surface. Adjacent layers differ in physical, chemical, and biological properties such as color, structure, texture, consistency, kinds and numbers of organisms present, and degree of acidity or alkalinity.
(xii) “Soil Order” is the broadest category of soil classification and is based on the general similarities of soil physical/chemical properties. The formation of soil by similar general genetic processes causes these similarities. The Soil Orders found in the United States are: Alfisol, Aridisol, Entisol, Histosol, Inceptisol, Mollisol, Oxisol, Spodosol, Ultisol, and Vertisol.
(xiii) “Soil series” is the basic unit of soil classification and is a subdivision of a family. A series consists of soils that were developed under comparable climatic and vegetational conditions. The soils comprising a series are essentially alike in all major profile characteristics except for the texture of the “A” horizon (i.e., the surface layer of soil).
(xiv) “Soil texture” is a classification of soils that is based on the relative proportions of the various soil separates present. The soil textural classes are: clay, sandy clay, silty clay, clay loam, silty clay loam, sandy clay loam, loam, silt loam, silt, sandy loam, loamy sand, and sand.
(3) Principle of the test method. (i) The extent of adsorption of a chemical onto sediment or soil is measured, using this test guideline, by equilibrating aqueous solutions containing different, but environmentally realistic, concentrations of the test chemical with a known quantity of sediment or soil. After equilibrium is reached, the distribution of the chemical between the water phase and the solid phase is quantitatively measured by a suitable analytical method. Then, sorption constants are calculated by using the Freundlich equation:
(ii) In order to estimate the environmental movement of the test chemical, the values K and l/n are compared with the values of other chemicals whose behavior in soil and sediment systems is well-documented in scientific literature.
(iii) The adsorption isotherm (AI) test has many desirable features. First, adsorption results are highly reproducible. The test provides excellent quantitative data readily amenable to statistical analyses. Also, it has relatively modest requirements for chemicals, soils, laboratory space, and equipment. It allows solution phase organic chemical determinations that are relatively uncomplicated. A chemical extraction-mass balance procedure to elicit information on chemical transformations occurring at colloid interfaces can be incorporated into this test. The ease of performing the isotherm test and mass balance will depend upon the physical/chemical properties of the test chemical and the availability of suitable analytical techniques to measure the chemical.
(iv) The papers by Aharonson and Kafkafi (1975) under paragraph (d)(1) of this section, Harvey (1974) under paragraph (d)(3) of this section, Murray (1975) under paragraph (d)(4) of this section, Saltzman (1972) under paragraph (d)(5) of this section, Weber (1971) under paragraph (d)(6) of this section, and Wu (1975) under paragraph (d)(7) of this section served as the basis for this section. The soil and colloid chemistry literature and the analytical chemistry literature substantiate the experimental conditions and procedures specified in this guideline as accepted, standard procedures.
(4) Applicability and specificity. The AI Test Guideline can be used to determine the soil and sediment adsorption potential of sparingly water soluble to infinitely soluble chemicals. In general, a chemical having a water solubility of less than 0.5 ppm need not be tested with soil as the solid phase, since the literature indicates that these chemicals are, in general, immobile in soils, see Goring and Hamaker (1972) under paragraph (d)(2) of this section. However, this does not preclude future soil adsorption/transformation testing of these chemicals if more refined data are needed for the assessment process.
(b) Test procedures – (1) Test conditions – (i) Special laboratory equipment. (A) Equilibrating solutions that contain, besides the test chemical, 0.01M calcium nitrate dissolved in sterilized, distilled-deionized H
(B) Containers shall be composed of material that (1) adsorb negligible amounts of test chemical, and (2) withstand high speed centrifugation. The volume of the container is not a major consideration; however, it is extremely important that the amount of soil or sediment and the solid/solution ratio used in the study result in minimal container headspace. It is also extremely important that the containers be sterilized before use.
(C) A 150 micron (100 mesh) stainless-steel or brass sieve.
(D) Drying oven, with circulating air, that can attain 100 °C.
(E) Vortex mixer or a comparable device.
(F) Rotary shaker or a comparable device.
(G) High speed temperature-controlled centrifuge capable of sedimenting particles greater than 0.5 micron from aqueous solution.
(ii) Temperature. The test procedure shall be performed at 23 ±5 °C.
(iii) Replications. Three replications of the experimental treatments shall be used.
(iv) Soil pretreatment. The following soil pretreatment steps shall be performed under the following conditions:
(A) Decrease the water content, air or oven-dry soils at or below 50 °C.
(B) Reduce aggregate size before and during sieving, crush and grind dried soil very gently.
(C) Eliminate microbial growth during the test period using a chemical or physical treatment that does not alter or minimally alters the soil surface properties.
(D) Sieve soils with a 100 mesh stainless-steel or brass sieve.
(E) Store all solutions and soils at temperatures between 0 and 5 °C.
(v) Sediment pretreatment. The following sediment pretreatment steps shall be performed under the following conditions:
(A) Decrease the H
(B) Eliminate microbial growth during the test period by using a chemical and/or physical treatment that does not alter or minimally alters the colloid surface’s properties.
(C) Store at temperatures between 0 and 5 °C.
(vi) Solid/solution ratio. The solid/solution ratio shall be equal to or greater than 1/10. If possible, the ratios should be equal to or greater than 1/5. The sediment or soil dry weight after drying for a 24-hour minimum at 90 °C is recommended for use as the weight of the solid for ratio and data calculations. If an insufficient amount of chemical remains in the water phase for quantification, the solid/solution ratio should be adjusted so that measurable amounts of the test chemical remain in solution.
(vii) Equilibration time. The equilibration time will depend upon the length of time needed for the parent chemical to attain an equilibrium distribution between the solid phase and the aqueous solution phase. The equilibration time shall be determined by the following procedure:
(A) Equilibrate one solution containing a known concentration of the test chemical with the sediment or soil in a solid/solution ratio equal to or greater than
(B) Measure the concentration of the chemical in the solution phase at frequent intervals during the equilibration period.
(C) Determine the equilibration time by plotting the measured concentration versus time of sampling; the equilibration time is the minimum period of time needed to establish a rate of change of solution concentration of 5 percent or less per 24 hours.
(viii) Centrifugation time. Calculate the centrifugation time, t
(ix) Storage of solutions. If the chemical analysis is delayed during the course of the experiment, store all solutions between 0 and 5 °C.
(x) Solvents for extraction. It is important that the solvent used to extract the chemical from the sediment or soil is reagent grade or better. Solvents shall contain no impurities which could interfere with the determination of the test compound.
(2) Test procedure – (i) Equilibration. Add six solutions containing different concentrations of the test chemical to at least one gram of each solid. The initial concentration of the test chemical in these solutions will depend on the affinity the chemical has for the sediment or soil. Therefore, after equilibrium is attained, it is extremely important that the highest concentration of the test chemical in the equilibrating solution does not exceed 10 ppm, is at least one order of magnitude greater than the lowest concentration reported, and does not exceed one half of its solubility.
(A) Immediately after the solutions are added to the solids, tightly cap the containers and vigorously agitate them for several minutes with a vortex mixture or similar device.
(B) Shake the containers throughout the equilibration period at a rate that suspends all solids in the solution phase.
(ii) Centrifugation. When the equilibration time has expired, centrifuge the containers for t
(iii) Chemical extraction. (A) After centrifugation, remove the supernatant aqueous phase from the solid-solution mixture.
(B) Extract the chemical adsorbed on the sediment or soil colloid surfaces with solvent.
(iv) Chemical analysis. Determine the amount of parent test chemical in the aqueous equilibrating solution and organic solvent extractions. Use any method or combination of methods suitable for the identification and quantitative detection of the parent test chemical.
(c) Reporting. Report the following information:
(1) Temperature at which the test was conducted.
(2) Detailed description of the analytical technique(s) used in the chemical extraction, recovery, and quantitative analysis of the parent chemical.
(3) Amount of parent test chemical applied, the amount recovered, and the percent recovered.
(4) Extent of adsorption by containers and the approach used to correct the data for adsorption by containers.
(5) The individual observations, the mean values, and graphical plots of x/m as a function of C
(6) The quantities K, n, and l/n.
(7) Soil information: Soil Order, series, texture, sampling location, horizon, general clay fraction mineralogy.
(8) Sediment information: sampling location, general clay fraction mineralogy.
(9) Sediment and soil physical-chemical properties: percent sand, silt, and clay (particle size analysis); percent organic matter; pH (1/1 solids/H
(10) The procedures used to determine the physical/chemical properties listed under paragraphs (c) (7) through (9) of this section.
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) Aharonson, N., Kafkafi, U. “Adsorption, mobility and persistence of thiabendazole and methyl 2-benzimidasole carbamate in soils,” Journal of Agricultural and Food Chemistry, 23:720-724 (1975).
(2) Goring, C.A.I., Hamaker, J.W., (eds). Organic Chemicals in the Soil Environment. Vol. I & II (New York: Marcel Dekker, Inc., 1972).
(3) Harvey, R.G. et al. “Soil adsorption and volatility of dinitroaniline herbicides,” Weed Science, 22:120-124 (1974).
(4) Murray, D.S. et al. “Comparative adsorption, desorption, and mobility of dipropetryn and prometryn in soil,” Journal of Agricultural and Food Chemistry, 23:578-581 (1973).
(5) Saltzman, S.L. et al. “Adsorption, desorption of parathion as affected by soil organic matter,” Journal of Agricultural and Food Chemistry, 20:1224-1226 (1972).
(6) Weber, J.B. “Model soil system, herbicide leaching, and sorption,” Weed Science, 19:145-160 (1971).
(7) Wu, C.H., et al. “Napropamide adsorption, desorption, and movement in soils,” Weed Science, 23:454-457 (1975).
Subpart D – Transformation Processes
§ 796.3100 Aerobic aquatic biodegradation.
(a) Introduction – (1) Purpose. (i) This Guideline is designed to develop data on the rate and extent of aerobic biodegradation that might occur when chemical substances are released to aquatic environments. A high biodegradability result in this test provides evidence that the test substance will be biodegradable in natural aerobic freshwater environments.
(ii) On the contrary, a low biodegradation result may have other causes than poor biodegradability of the test substance. Inhibition of the microbial inoculum by the test substance at the test concentration may be observed. In such cases, further work is needed to assess the aerobic aquatic biodegradability and to determine the concentrations at which toxic effects are evident. An estimate of the expected environmental concentration will help to put toxic effects into perspective.
(2) Definitions. (i) “Adaptation” is the process by which a substance induces the synthesis of any degradative enzymes necessary to catalyze the transformation of that substance.
(ii) “Ultimate Biodegradability” is the breakdown of an organic compound to CO
(iii) “Ready Biodegradability” is an expression used to describe those substances which, in certain biodegradation test procedures, produce positive results that are unequivocal and which lead to the reasonable assumption that the substance will undergo rapid and ultimate biodegradation in aerobic aquatic environments.
(3) Principle of the test method. This Guideline method is based on the method described by William Gledhill (1975) under paragraph (d)(1) of this section. The method consists of a 2-week inoculum buildup period during which soil and sewage microorganisms are provided the opportunity to adapt to the test compound. This inoculum is added to a specially equipped Erlenmeyer flask containing a defined medium with test substance. A reservoir holding barium hydroxide solution is suspended in the test flask. After inoculation, the test flasks are sparged with CO
(4) Prerequisites. The total organic carbon (TOC) content of the test substance shall be calculated or, if this is not possible, analyzed, to enable the percent of theoretical yield of carbon dioxide and percent of DOC loss to be calculated.
(5) Guideline information. (i) Information on the relative proportions of the major components of the test substance will be useful in interpreting the results obtained, particularly in those cases where the result lies close to a “pass level.”
(ii) Information on the toxicity of the chemical may be useful in the interpretation of low results and in the selection of appropriate test concentrations.
(6) Reference substances. Where investigating a chemical substance, reference compounds may be useful and an inventory of suitable reference compounds needs to be identified. In order to check the activity of the inoculum the use of a reference compound is desirable. Aniline, sodium citrate, dextrose, phthalic acid and trimellitic acid will exhibit ultimate biodegradation under the conditions of this Test Guideline method. These reference substances must yield 60 percent of theoretical maximum CO
(7) Reproducibility. The reproducibility of the method has not yet been determined; however it is believed to be appropriate for a screening test which has solely an acceptance but no rejective function.
(8) Sensitivity. The sensitivity of the method is determined by the ability to measure the endogenous CO
(9) Possibility of standardization. This possibility exists. The major difficulty is to standardize the inoculum in such a way that interlaboratory reproducibility is ensured.
(10) Possibility of automation. None at present, although parts of the analyses may be automated.
(b) Test procedures – (1) Preparations – (i) Apparatus. The shake flask apparatus under the following Figure 1 contains 10 mL of 0.2N Ba(OH)
(ii) Reagents and stock solutions. (A) Stock solutions, I, II, and III under the following Table 1.
(B) Yeast extract.
(C) Vitamin-free casamino acids.
(D) 70 percent O
(E) 0.2N Ba(OH)
(F) 0.1 N HCl.
(G) 20 percent H
(H) Phenolphthalein.
(I) Dilution water – distilled, deionized water (DIW).
(iii) Soil inoculum. A fresh sample of an organically rich soil is used as the inoculum in the ultimate biodegradation test. Soil is collected, prepared, and stored according to the recommendations of Pramer and Bartha (1972) under paragraph (d)(2) of this section. The soil surface is cleared of litter and a soil sample is obtained 10 to 20 cm below the surface. The sample is screened through a sieve with 2 to 5 mm openings and stored in a polyethylene bag at 2 to 4 °C for not more than 30 days prior to use. The soil is never allowed to air-dry, and shall not be frozen during storage.
Table 1 – Medium Employed for Assay of CO
| Solution 1 | Compound | Stock Solution Conc. (g/L) |
|---|---|---|
| I | NH | 35 |
| KNO | 15 | |
| K | 750 | |
| NaH | 25 | |
| II 2 | KCl | 10 |
| MgSO | 20 | |
| FeSO | 1 | |
| III | CaCl | 5 |
| ZnCl | 0.05 | |
| MnCl | 0.5 | |
| CuCl | 0.05 | |
| CoCl | 0.001 | |
| H | 0.001 | |
| MoO | 0.0004 |
1 = Each liter of test medium contains 1 mL of each solution.
2 = Final pH is adjusted to 3.0 with 0.10 N HCl.
(iv) Acclimation Medium. Acclimation medium is prepared by adding, for each liter of distilled, deionized water (DIW): 1 mL each of solutions I, II, and III in Table 1 in paragraph (b)(1)(iii) of this section, 1.0 gm of soil inoculum (prepared according to paragraph (b)(1)(iii) of this section), 2.0 mL of aerated mixed liquor (obtained from an activated sludge treatment plant not more than 2 days prior to commencing the acclimation phase, and stored in the interim at 4 °C) and 50 mL raw domestic influent sewage. This medium is mixed for 15 minutes and filtered through a glass wool plug in a glass funnel. The filtrate is permitted to stand for 1 hour, refiltered through glass wool, and supplemented with 25 mg/L each of Difco vitamin-free casamino acids and yeast extract. Appropriate volumes are added to 2-liter Erlenmeyer flasks. Test compounds are added incrementally during the acclimation period at concentrations equivalent to 4, 8, and 8 mg/L carbon on days 0, 7, and 11, respectively. On day 14, the medium is refiltered through glass wool prior to use in the test. For evaluating the biodegradability of a series of functionally or structurally related chemicals, media from all inoculum flasks may be combined before final filtration.
(2) Procedures. (i) Inoculum (100 mL of acclimation medium) is added to 900 mL DIW containing 1 mL each of solutions I, II, and III in Table 1 under paragraph (b)(1)(iii) of this section in a 2-liter Erlenmeyer flask. Test compound equivalent to 10 mg/liter carbon is added to each of the replicate flasks containing the test medium. Ten mL of 0.2 N Ba (OH)
(ii) For each set of experiments, each test substance shall be tested in triplicate.
(iii) For each set of experiments, one or two reference compounds are included to assess the microbial activity of the test medium. Duplicate reference flasks are prepared by adding reference compound equivalent to 10 mg/liter carbon to each of two flasks containing the test medium. Reference compounds which are positive for ultimate biodegradability include: sodium citrate, dextrose, phthalic acid, trimellitic acid, and aniline.
(iv) For each test set, triplicate controls receiving inoculated medium and no test compound, plus all test and reference flasks, are analyzed for CO
(v) A test system containing a growth inhibitor should be established as a control for each substance tested for biodegradation by this method. That inhibited system must contain the same amount of water, mineral nutrients, inoculum, and test substance used in the uninhibited test systems, plus 50 mg/L mercuric chloride (HgCl
(vi) Flasks shall be incubated in the dark to minimize both photochemical reactions and algal growth. Appropriate sterile controls or controls containing a metabolic inhibitor, such as 50 mg/1 HgCl
(3) Analytical measurements. The quantity of CO
(c) Data and reporting – (1) Treatment of results. (i) Test compound (10 mg carbon) is theoretically converted to 0.833 mmol CO
(ii) The cumulative percent CO
(iii) The percent DOC disappearance from the test compound is calculated from the following equation:
(iv) The difference between the amount of 0.1 N HCl used for the Ba(OH)
(v) CO
(vi) Inhibition by the test compound is indicated by lower CO
(vii) The use of
(2) Test report. (i) For each test and reference compound, the following data shall be reported.
(ii) Information on the inoculum, including source, collection date, handling, storage and adaptation possibilities (i.e., that the inoculum might have been exposed to the test substance either before or after collection and prior to use in the test).
(iii) Results from each test, reference, inhibited (with HgCl
(iv) Average cumulative percent theoretical CO
(v) Dissolved organic carbon due to test compound at each sampling time (DTF-DCF).
(vi) Average percent DOC removal at each sampling time.
(vii) Twenty-eight day standard deviation for percent CO
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) Gledhill, W.E. “Screening Test for Assessment of Ultimate Biodegradability: Linear Alkyl Benzene Sulfonate,” Applied Microbiology, 30:922-929 (1975).
(2) Pramer, D., Bartha, R. “Preparation and Processing of Soil Samples for Biodegradation Testing,” Environmental Letters, 2:217-224 (1972).
§ 796.3500 Hydrolysis as a function of pH at 25 °C.
(a) Introduction – (1) Background and purpose. (i) Water is one of the most widely distributed substances in the environment. It covers a large portion of the earth’s surface as oceans, rivers, and lakes. The soil also contains water, as does the atmosphere in the form of water vapor. As a result of this ubiquitousness, chemicals introduced into the environment almost always come into contact with aqueous media. Certain classes of these chemicals, upon such contact, can undergo hydrolysis, which is one of the most common reactions controlling chemical stability and is, therefore, one of the main chemical degradation paths of these substances in the environment.
(ii) Since hydrolysis can be such an important degradation path for certain classes of chemicals, it is necessary, in assessing the fate of these chemicals in the environment, to know whether, at what rate, and under what conditions a substance will hydrolyze. Some of these reactions can occur so rapidly that there may be greater concern about the products of the transformation than about the parent compounds. In other cases, a substance will be resistant to hydrolysis under typical environmental conditions, while, in still other instances, the substance may have an intermediate stability that can result in the necessity for an assessment of both the original compound and its transformation products. The importance of transformation of chemicals via hydrolysis in aqueous media in the environment can be determined quantitatively from data on hydrolysis rate constants. This hydrolysis Test Guideline represents a test to allow one to determine rates of hydrolysis at any pH of environmental concern at 25 °C.
(2) Definitions and units. (i) “Hydrolysis” is defined as the reaction of an organic chemical with water, such that one or more bonds are broken and the reaction products of the transformation incorporate the elements of water (H
(ii) “Elimination” is defined in this Test Guideline to be a reaction of an organic chemical (RX) in water in which the X group is lost. These reactions generally follow the same type of rate laws that hydrolysis reactions follow and, thus, are also covered in this Test Guideline.
(iii) A “first-order reaction” is defined as a reaction in which the rate of disappearance of the chemical substance being tested is directly proportional to the concentration of the chemical substance and is not a function of the concentrations of any other substances present in the reaction mixture.
(iv) The “half-life” of a chemical is defined as the time required for the concentration of the chemical substance being tested to be reduced to one-half its initial value.
(v) “Hydrolysis” refers to a reaction of an organic chemical with water such that one or more bonds are broken and the reaction products incorporate the elements of water (H
(A) Another result of hydrolysis can be the incorporation of both H and OH in a single product. An example of this is the hydrolysis of epoxides, which can be represented by
(B) The hydrolysis reaction can be catalyzed by acidic or basic species, including OH
(C) At a given pH, Equation 2 under paragraph (a)(2)(v)(B) of this section contains three unknowns, k
(D) The equations under paragraph (a)(2) of this section apply whether the test chemical has one or more hydrolyzable groups. In the latter case, the rate may be written as:
(3) Principle of the test method. Procedures described in this section enable sponsors to obtain quantitative information on hydrolysis rates through a determination of hydrolysis rate constants and half-lives of chemicals at pH 3.00, 7.00, and 11.00 at 25 °C. The three measured rate constants are used to determine the acidic, basic, and neutral rate constants associated with a hydrolytic reaction. The latter constants can then be employed in determining the hydrolysis rates of chemicals at any pH of environmental concern at 25 °C.
(4) Applicability and specificity. There are several different common classes of organic chemicals that are subject to hydrolysis transformation, including esters, amides, lactones, carbamates, organophosphates, and alkyl halides. Processes other than nucleophilic displacement by water can also take place. Among these are elimination reactions that exhibit behavior similar to hydrolysis and, therefore, are also covered in this section.
(b) Test procedures – (1) Test conditions – (i) Special laboratory equipment. (A) A thermostatic bath that can be maintained at a temperature of 25 ±1 °C.
(B) A pH meter that can resolve differences of 0.05 pH units or less.
(C) Stoppered volumetric flasks (no grease) or glass ampoules that can be sealed.
(ii) Purity of water. Reagent-grade water (e.g., water meeting ASTM Type IIA standards or an equivalent grade) shall be used to minimize biodegradation. ASTM Type IIA water is described in ASTM D 1193-77 (Reapproved 1983), “Standard Specification for Reagent Water.” ASTM D 1193-77 (Reapproved 1983) is available for inspection at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. This incorporation by reference was approved by the Director of the Office of the Federal Register. This material is incorporated as it exists on the date of approval and a notice of any change in this material will be published in the
(iii) Sterilization. All glassware shall be sterilized. Aseptic conditions shall be used in the preparation of all solutions and in carrying out all hydrolysis experiments to eliminate or minimize biodegradation. Glassware can be sterilized in an autoclave or by any other suitable method.
(iv) Precautions for volatility. If the chemical is volatile the reaction vessels shall be almost completely filled and sealed.
(v) Temperature controls. All hydrolysis reactions shall be carried out at 25 °C (±1 °C) and with the temperature controlled to ±0.1 °C.
(vi) pH conditions. It is recommended that all hydrolysis experiments be performed at pH 3.00, 7.00, and 11.00 ±0.05 using the appropriate buffers described in paragraph (b)(2)(i)(A) of this section.
(vii) Concentration of solutions of chemical substances. The concentration of the test chemical shall be less than one-half the chemical’s solubility in water but not greater than 10
(viii) Effect of acidic and basic groups. Complications can arise upon measuring the rate of hydrolysis of chemicals that reversibly ionize or are protonated in the pH range 3.00 to 11.00. Therefore, for these chemicals, it is recommended that these hydrolysis tests be performed at pH 5.00, 7.00, and 900 ±0.05 using the appropriate buffers described in paragraphs (b)(2)(i) (A) and (B) of this section. If a test chemical reversibly ionizes or protonates in the pH range 5.00 to 9.00, then it is recommended that additional hydrolysis tests should be carried out at pH 6.00 and 8.00 ±0.05 using the buffers described in paragraph (b)(2)(i)(B) of this section.
(ix) Buffer catalysis. For certain chemicals, buffers may catalyze the hydrolysis reaction. If this is suspected, hydrolysis rate determination shall be carried out with the appropriate buffers and the same experiments repeated at buffer concentrations lowered by at least a factor of five. If the hydrolysis reaction produces a change of greater than 0.05 pH units in the lower concentration buffers at the end of the measurement time, the test chemical concentrations also shall be lowered by at least a factor of five. Alternatively, test chemical concentrations and buffer concentrations may both be lowered simultaneously by a factor of five. A sufficient criterion for minimization of buffer catalysis is an observed equality in the hydrolysis rate constant for two different solutions differing in buffer or test chemical concentration by a factor of five.
(x) Photosensitive chemicals. The solution absorption spectrum can be employed to determine whether a particular chemical is potentially subject to photolytic transformation upon exposure to light. For chemicals that absorb light of wavelengths greater than 290 nm, the hydrolysis experiment shall be carried out in the dark, under amber or red safelights, in amber or red glassware, or employing other suitable methods for preventing photolysis. The absorption spectrum of the chemical in aqueous solution can be measured under § 796.1050.
(xi) Chemical analysis of solutions. In determining the concentrations of the test chemicals in solution, any suitable analytical method may be employed, although methods which are specific for the compound to be tested are preferred. Chromatographic methods are recommended because of their compound specificity in analyzing the parent chemical without interferences from impurities. Whenever practicable, the chosen analytical method should have a precision within ±5 percent.
(2) Preparation – (i) Reagents and solutions – (A) Buffer solutions. Prepare buffer solutions using reagent-grade chemicals and reagent-grade water as follows:
(1) pH 3.00: use 250 mL of 0.100M potassium hydrogen phthalate; 111 mL of 0.100M hydrochloric acid; and adjust volume to 500 mL with reagent-grade water.
(2) pH 7.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 145 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade water.
(3) pH 11.00: use 250 mL of 0.0500M sodium bicarbonate; 113 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade water.
(B) Additional buffer solutions. For chemicals that ionize or are protonated as discussed in paragraph (b)(1)(viii) of this section, prepare buffers using reagent-grade water and reagent-grade chemicals as follows:
(1) pH 5.00: use 250 mL of 0.100M potassium hydrogen phthalate; 113 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade water.
(2) pH 6.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 28 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade water.
(3) pH 8.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 234 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade water.
(4) pH 9.00: use 250 mL of 0.0250M borax (Na
(C) Adjustment of buffer concentrations. (1) The concentrations of all the above buffer solutions are the maximum concentration to be employed in carrying out hydrolysis measurements. If the initial concentration of the test chemical is less than 10
(2) Check the pH of all buffer solutions with a pH meter at 25 °C and adjust the pH to the proper value, if necessary.
(D) Preparation of test solution. (1) If the test chemical is readily soluble in water, prepare an aqueous solution of the chemical in the appropriate buffer and determine the concentration of the chemical. Alternatively, a solution of the chemical in water may be prepared and added to an appropriate buffer solution and the concentration of the chemical then determined. In the latter case, the aliquot shall be small enough so that the concentration of the buffer in the final solution and the pH of the solution remain essentially unchanged. Do not employ heat in dissolving the chemical. The final concentration shall not be greater than one-half the chemical’s solubility in water and not greater than 10
(2) If the test chemical is too insoluble in pure water to permit reasonable handling and analytical procedures, it is recommended that the chemical be dissolved in reagent-grade acetonitrile and buffer solution and then added to an aliquot of the acetonitrile solution. Do not employ heat to dissolve the chemical in acetonitrile. The final concentration of the test chemical shall not be greater than one-half the chemical’s solubility in water and not greater than 10
(3) Performance of the test. Carry out all hydrolysis experiments by employing one of the procedures described in this paragraph. Prepare the test solutions as described in paragraph (b)(2)(i) of this section at pH 3.00, 7.00, and 11.00 ±0.05, and determine the initial test chemical concentration (C
(i) Procedure 1. Analyze each test solution at regular intervals to provide a minimum of six measurements with the extent of hydrolysis between 20 to 70 percent. Rates should be rapid enough so that 60 to 70 percent of the chemical is hydrolyzed in 672 hours.
(ii) Procedure 2. If the reaction is too slow to conveniently follow hydrolysis to high conversion in 672 hours but still rapid enough to attain at least 20 percent conversion, take 15 to 20 time points at regular intervals after 10 percent conversion is attained.
(iii) Procedure 3. (A) If chemical hydrolysis is less than 20 percent after 672 hours, determine the concentration (C) after this time period.
(B) If the pH at the end of concentration measurements employing any of the above three procedures has changed by more than 0.05 units from the initial pH, repeat the experiment using a solution having a test chemical concentration lowered sufficiently to keep the pH variation within 0.05 pH units.
(iv) Analytical methodology. Select an analytical method that is most applicable to the analysis of the specific chemical being tested under paragraph (b)(1)(xi) of this section.
(c) Data and reporting – (1) Treatment of results. (i) If Procedure 1 or 2 were employed in making concentration measurements, use a linear regression analysis with Equation 4 under paragraph (a)(2)(v)(B) of this section to calculate k
(ii) If Procedure 3 was employed in making rate measurements, use the mean initial concentration (C
(iii) For each set of three concentration replicates, calculate the mean value of C and the standard deviation.
(iv) For test chemicals that are not ionized or protonated between pH 3 and 11, calculate k
(2) Specific analytical and recovery procedures. (i) Provide a detailed description or reference for the analytical procedure used, including the calibration data and precision.
(ii) If extraction methods were used to separate the solute from the aqueous solution, provide a description of the extraction method as well as the recovery data.
(3) Test data report. (i) For Procedures 1 and 2, report k
(ii) For Procedure 3, report k
(iii) If, after 672 hours, the concentration (C) is the same as the initial concentration (C
PART 797 – ENVIRONMENTAL EFFECTS TESTING GUIDELINES
Subpart A [Reserved]
Subpart B – Aquatic Guidelines
§ 797.1050 Algal acute toxicity test.
(a) Purpose. The guideline in this section is intended for use in developing data on the acute toxicity of chemical substances and mixtures (“chemicals”) subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes test procedures and conditions using freshwater and marine algae to develop data on the phytotoxicity of chemicals. The United States Environmental Protection Agency (U.S. EPA) will use data from these tests in assessing the hazard of a chemical to the environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA) and the definitions in part 792 – Good Laboratory Practice Standards of this chapter apply to this test guideline. The following definitions also apply to this guideline:
(1) Algicidal means having the property of killing algae.
(2) Algistatic means having the property of inhibiting algal growth.
(3) ECx means the experimentally derived chemical concentration that is calculated to effect X percent of the test criterion.
(4) Growth means a relative measure of the viability of an algal population based on the number and/or weight of algal cells per volume of nutrient medium or test solution in a specified period of time.
(5) Static system means a test container in which the test solution is not renewed during the period of the test.
(c) Test procedures – (1) Summary of the test. (i) In preparation for the test, fill test containers with appropriate volumes of nutrient medium and/or test solution. Start the test by introducing algae into the test and control containers in the growth chambers. Environmental conditions within the growth chambers are established at predetermined limits.
(ii) At the end of 96 hours enumerate the algal cells in all containers to determine inhibition or stimulation of growth in test containers compared to controls. Use data to define the concentration-response curve, and calculate the EC
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted to determine:
(A) If definitive testing is necessary.
(B) Test chemical concentrations for the definitive test.
(ii) Algae are exposed to a widely spaced (e.g., log interval) chemical concentration series. The lowest value in the series, exclusive of controls, should be at the chemical’s detection limit. The upper value, for water soluble compounds, should be the saturation concentration. No replicates are required; and nominal concentrations of the chemical are acceptable unless definitive testing is not required.
(iii) The test is performed once for each of the recommended algal species or selected alternates. Test chambers should contain equal volumes of test solution and approximately 1 × 10
(iv) Definitive testing is not necessary if the highest chemical concentration tested (water saturation concentration or 1000 mg/l) results in less than a 50 percent reduction in growth or if the lowest concentration tested (analytical detection limit) results in greater than a 50 percent reduction in growth.
(4) Definitive test. (i) The purpose of the definitive test is to determine the concentration response curves, the EC
(ii) Algae should be exposed to five or more concentrations of the test chemical in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). Algae shall be placed in a minimum of three replicate test containers for each concentration of test chemical and control. More than three replicates may be required to provide sufficient quantities of test solution for determination of test substance concentration at the end of the test. Each test chamber should contain equal volumes of test solution and approximately 1 × 10
(iii) Every test shall include a control consisting of the same nutrient medium, conditions, procedures, and algae from the same culture, except that none of the test substance is added. If a carrier is present in any of the test chambers, a separate carrier control is required.
(iv) The test begins when algae from 5- to 10-day-old stock cultures are placed in the test chambers containing test solutions having the appropriate concentrations of the test substance. Algal growth in controls should reach the logarithmic growth phase by 96 hours. If logarithmic growth cannot be demonstrated, the test shall be repeated. At the end of 24, 48, 72, and 96 hours the algal growth response (number or weight of algal cells/ml) in all test containers and controls shall be determined by an indirect (spectrophotometry, electronic cell counters, dry weight, etc.) or a direct (actual microscopic cell count) method. Indirect methods shall be calibrated by a direct microscopic count. The percentage inhibition or stimulation of growth for each concentration, EC
(v) At the end of the definitive test, the following additional analyses of algal growth response shall be performed:
(A) Determine whether the altered growth response between controls and test algae was due to a change in relative cell numbers, cell sizes or both. Also note any unusual cell shapes, color differences, flocculations, adherence of algae to test containers, or aggregation of algal cells.
(B) In test concentrations where growth is maximally inhibited, algistatic effects may be differentiated from algicidal effects by the following two methods for Skeletonema and by the second method for Selenastrum.
(1) Add 0.5 ml of a 0.1 percent solution (weight/volume) of Evans blue stain to a 1 milliliter aliquot of algae from a control container and to a 1 milliliter aliquot of algae from the test container having the lowest concentration of test chemical which completely inhibited algal growth (if algal growth was not completely inhibited, select an aliquot of algae for staining from the test container having the highest concentration of test chemical which inhibited algal growth). Wait 10 to 30 minutes, examine microscopically, and determine the percent of the cells which stain blue (indicating cell mortality). A staining control shall be performed concurrently using heat-killed or formaldehyde-preserved algal cells; 100 percent of these cells shall stain blue.
(2) Remove 0.5 ml aliquots of test solution containing growth-inhibited algae from each replicate test container having the concentration of test substance evaluated in paragraph (c)(4)(v)(B)(1) of this section. Combine these aliquots into a new test container and add a sufficient volume of fresh nutrient medium to dilute the test chemical to a concentration which does not affect growth. Incubate this subculture under the environmental conditions used in the definitive test for a period of up to 9 days, and observe for algal growth to determine if the algistatic effect noted after the 96-hour test is reversible. This subculture test may be discontinued as soon as growth occurs.
(5) [Reserved]
(6) Analytical measurements – (i) Chemical. (A) Glass distilled or deionized water shall be used in the preparation of the nutrient medium. The pH of the test solution shall be measured in the control and test containers at the beginning and at the end of the definitive test. The concentration of test chemical in the test containers shall be determined at the beginning and end of the definitive test by standard analytical methods which have been validated prior to the test. An analytical method is unacceptable if likely degradation products of the chemical, such as hydrolysis and oxidation products, give positive or negative interference.
(B) At the end of the test and after aliquots have been removed for algal growth-response determinations, microscopic examination, mortal staining, or subculturing, the replicate test containers for each chemical concentration may be pooled into one sample. An aliquot of the pooled sample may then be taken and the concentration of test chemical determined. In addition, the concentration of test chemical associated with the algae alone should be determined. Separate and concentrate the algal cells from the test solution by centrifuging or filtering the remaining pooled sample and measure the test substance concentration in the algal-cell concentrate.
(ii) Numerical. Algal growth response (as percent of inhibition or stimulation in the test solutions compared to the controls) is calculated at the end of the test. Mean and standard deviation should be calculated and plotted for each treatment and control. Appropriate statistical analyses should provide a goodness-of-fit determination for the concentration response curves. The concentration response curves are plotted using the mean measured test solution concentrations obtained at the end of the test.
(d) Test conditions – (1) Test species. Species of algae recommended as test organisms for this test are the freshwater green alga, Selenastrum capricornutum, and the marine diatom, Skeletonema costatum. Algae to be used in acute toxicity tests may be initially obtained from commercial sources and subsequently cultured using sterile technique. Toxicity testing shall not be performed until algal cultures are shown to be actively growing (i.e., capable of logarithmic growth within the test period) in at least 2 subcultures lasting 7 days each prior to the start of the definitive test. All algae used for a particular test shall be from the same source and the same stock culture. Test algae shall not have been used in a previous test, either in a treatment or a control.
(2) Facilities – (i) General. (A) Facilities needed to perform this test include: a growth chamber or a controlled environment room that can hold the test containers and will maintain the air temperature, lighting intensity and photoperiod specified in this test guideline; apparatus for culturing and enumerating algae; a source of distilled and/or deionized water; and apparatus for carrying out analyses of the test chemical.
(B) Disposal facilities should be adequate to accommodate spent glassware, algae and test solutions at the end of the test and any bench covering, lab clothing, or other contaminated materials.
(ii) Test containers. Erlenmeyer flasks should be used for test containers. The flasks may be of any volume between 125 and 500 ml as long as the same size is used throughout a test and the test solution volume does not exceed 50 percent of the flask volume.
(iii) Cleaning and sterilization. New test containers may contain substances which inhibit growth of algae. They shall therefore be cleaned thoroughly and used several times to culture algae before being used in toxicity testing. All glassware used in algal culturing or testing shall be cleaned and sterilized prior to use according to standard good laboratory practices.
(iv) Conditioning. Test containers should be conditioned by a rinse with the appropriate test solutions prior to the start of the test. Decant and add fresh test solutions after an appropriate conditioning period for the test chemical.
(v) Nutrient medium. (A) Formulation and sterilization of nutrient medium used for algal culture and preparation of test solutions should conform to those currently recommended by the U.S. EPA for freshwater and marine algal bioassays. No chelating agents are to be included in the nutrient medium used for test solution preparation. Nutrient medium should be freshly prepared for algal testing and may be dispensed in appropriate volumes in test containers and sterilized by autoclaving or filtration. The pH of the nutrient medium shall be 7.5 (±0.1) for Selenastrum and 8.1 (±0.1) for Skeletonema at the start of the test and may be adjusted prior to test chemical addition with 0.1N NaOH or HC1.
(B) Dilution water used for preparation of nutrient medium and test solutions should be filtered, deionized or glass distilled. Saltwater for marine algal nutrient medium and test solutions should be prepared by adding a commercial, synthetic, sea salt formulation or a modified synthetic seawater formulation to distilled/deionized water to a concentration of 30 parts per thousand.
(vi) Carriers. Nutrient medium shall be used in making stock solutions of the test chemical. If a carrier other than nutrient medium is absolutely necessary to dissolve the chemical, the volume used shall not exceed the minimum volume necessary to dissolve or suspend the chemical in the test solution.
(3) Test parameters. (i) The test temperature shall be 24 °C for Selenastrum and 20 °C for Skeletonema. Excursions from the test temperature shall be no greater than ±2 °C. Temperature should be recorded hourly during the test.
(ii) Test chambers containing Selenastrum shall be illuminated continuously and those containing Skeletonema shall be provided a 14-hour light and 10-hour dark photoperiod with a 30 minute transition period under fluorescent lamps providing 300 ±25 uEin/m
(iii) Stock algal cultures should be shaken twice daily by hand. Test containers shall be placed on a rotary shaking apparatus and oscillated at approximately 100 cycles/minute for Selenastrum and at approximately 60 cycles/minute for Skeletonema during the test. The rate of oscillation should be determined at least once daily during testing.
(iv) The pH of nutrient medium in which algae are subcultured shall be 7.5 (±0.1) for Selenastrum and 8.1 (±0.1) for Skeletonema, and is not adjusted after the addition of the algae. The pH of all test solutions shall be measured at the beginning and end of the test.
(v) Light intensity shall be monitored at least daily during the test at the level of the test solution.
(e) Reporting. The sponsor shall submit to the EPA all data developed by the test that are suggestive or predictive of acute phytotoxicity. In addition to the general reporting requirements prescribed in part 792 – Good Laboratory Practice Standards of this Chapter, the following shall be reported:
(1) Detailed information about the test organisms, including the scientific name, method of verification, and source.
(2) A description of the test chambers and containers, the volumes of solution in the containers, the way the test was begun (e.g., conditioning, test substance additions, etc.), the number of replicates, the temperature, the lighting, and method of incubation, oscillation rates, and type of apparatus.
(3) The concentration of the test chemical in the control and in each treatment at the end of the test and the pH of the solutions.
(4) The number of algal cells per milliliter in each treatment and control and the method used to derive these values at the beginning, 24, 48, and 72 hours, and end of the test; the percentage of inhibition or stimulation of growth relative to controls; and other adverse effect in the control and in each treatment.
(5) The 96-hour EC
(6) Methods and data records of all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks.
(7) The results of any optional analyses such as: Microscopic appearance of algae, size or color changes, percent mortality of cells and the fate of subcultured cells, the concentration of test substance associated with algae and test solution supernate or filtrate.
(8) If the range-finding test showed that the highest concentration of the chemical tested (not less than 1000 mg/l or saturation concentration) had no effect on the algae, report the results and concentration and a statement that the chemical is of minimum phytotoxic concern.
(9) If the range-finding test showed greater than a 50 percent inhibition of algal growth at a test concentration below the analytical detection limit, report the results, concentration, and a statement that the chemical is phytotoxic below the analytical detection limit.
§ 797.1300 Daphnid acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data on the acute toxicity of chemical substances and mixtures (“chemicals”) subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes an acute toxicity test in which daphnids (Daphnia magna or D. pulex) are exposed to a chemical in static and flow-through systems. The United States Environmental Protection Agency will use data from this test in assessing the hazard a chemical may present in the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA) and part 792 – Good Laboratory Practice Standards of this chapter apply to this test guideline. In addition, the following definitions apply to this guideline:
(1) Brood stock means the animals which are cultured to produce test organisms through reproduction.
(2) EC
(3) Ephippium means a resting egg which develops under the carapace in response to stress conditions in daphnids.
(4) Flow-through means a continuous or an intermittent passage of test solution or dilution water through a test chamber or culture tank with no recycling.
(5) Immobilization means the lack of movement by the test organisms except for minor activity of the appendages.
(6) Loading means the ratio of daphnid biomass (grams, wet weight) to the volume (liters) of test solution in a test chamber at a point in time, or passing through the test chamber during a specific interval.
(7) Static system means a test system in which the test solution and test organisms are placed in the test chamber and kept there for the duration of the test without renewal of the test solution.
(c) Test procedures – (1) Summary of the test. (i) Test chambers are filled with appropriate volumes of dilution water. In the flow-through test, the flow of dilution water through each chamber is adjusted to the rate desired. The test chemical is introduced into each treatment chamber. The addition of test chemical in the flow-through system is conducted at a rate which is sufficient to establish and maintain the desired concentration in the test chamber. The test is started within 30 minutes after the test chemical has been added and uniformly distributed in static test chambers or after the concentration of test chemical in each flow-through test chamber reaches the prescribed level and remains stable. At the initiation of the test, daphnids which have been cultured and acclimated in accordance with the test design are randomly placed into the test chambers. Daphnids in the test chambers are observed periodically during the test, the immobile daphnids removed, and the findings recorded.
(ii) Dissolved oxygen concentration, pH, temperature, the concentration of test chemical and other water quality parameters are measured at specified intervals in selected test chambers. Data are collected during the test to develop concentration-response curves and determine EC
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted to establish test solution concentrations for the definitive test.
(ii) The daphnids should be exposed to a series of widely spaced concentrations of the test chemical (e.g., 1, 10, 100 mg/1, etc.), usually under static conditions.
(iii) A minimum of five daphnids should be exposed to each concentration of test chemical for a period of 48 hours. The exposure period may be shortened if data suitable for the purpose of the range-finding test can be obtained in less time. No replicates are required and nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to determine the concentration-response curves and the 24- and 48-hour EC
(ii) A minimum of 20 daphnids per concentration shall be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). An equal number of daphnids shall be placed in two or more replicates. If solvents, solubilizing agents or emulsifiers have to be used, they shall be commonly used carriers and shall not possess a synergistic or antagonistic effect on the toxicity of the test chemical. The concentration of solvent should not exceed 0.1 mg/l. The concentration ranges shall be selected to determine the concentration-response curves and EC
(iii) Every test shall include controls consisting of the same dilution water, conditions, procedures and daphnids from the same population (culture container), except that none of the chemical is added.
(iv) The dissolved oxygen concentration, temperature and pH shall be measured at the beginning and end of the test in each chamber.
(v) The test duration is 48 hours. The test is unacceptable if more than 10 percent of the control organisms are immobilized during the 48-hour test period. Each test chamber shall be checked for immobilized daphnids at 24 and 48 hours after the beginning of the test. Concentration-response curves and 24-hour and 48-hour EC
(vi) In addition to immobility, any abnormal behavior or appearance shall also be reported.
(vii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner or in a way in which appropriate statistical analyses can be used to determine the variation due to placement.
(viii) The concentration of the test chemical in the chambers should be measured as often as is feasible during the test. In the static test the concentration of test chemical shall be measured, at a minimum, at the beginning of the test and at the end of the test in each test chamber. In the flow-through test the concentration of test chemical shall be measured at a minimum:
(A) In each chamber at the beginning of the test and at 48 hours after the start of the test;
(B) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system.
(5) [Reserved]
(6) Analytical measurements. (i) Test chemical. Deionized water should be used in making stock solutions of the test chemical. Standard analytical methods should be used whenever available in performing the analyses. The analytical method used to measure the amount of test chemical in a sample shall be validated before beginning the test by appropriate laboratory practices. Any analytical method is not acceptable if likely degradation products of the test chemical, such as hydrolysis and oxidation products, give positive or negative interferences which cannot be systematically identified and corrected mathematically.
(ii) Numerical. The number of immobilized daphnids shall be counted during each definitive test. Appropriate statistical analyses should provide a goodness-of-fit determination for the concentration-response curves. A 24- and 48-hour EC
(d) Test conditions – (1) Test species – (i) Selection. (A) The cladocerans, Daphnia magna or D. pulex, are the test species to be used in this test. Either species may be used for testing of a particular chemical. The species identity of the test organisms should be verified using appropriate systematic keys. First instar daphnids, ≤24 hours old, are to be used to start the test.
(B) Daphnids to be used in acute toxicity tests should be cultured at the test facility. Records should be kept regarding the source of the initial stock and culturing techniques. All organisms used for a particular test shall have originated from the same culture population.
(C) Daphnids shall not be used for a test (1) if cultures contain ephippia; (2) if adults in the cultures do not produce young before day 12; (3) if more than 20 percent of the culture stock die during the 2 days preceding the test; (4) if adults in the culture do not produce an average of at least 3 young per adult per day over the 7-day period prior to the test and (5) if daphnids have been used in any portion of a previous test, either in a treatment or in a control.
(ii) Acclimation. (A) Brood daphnids shall be maintained in 100-percent dilution water at the test temperature for at least 48 hours prior to the start of the test. This is easily accomplished by culturing them in the dilution water at the test temperature. During production of neonates, daphnids should not be fed.
(B) During culturing and acclimation to the dilution water, daphnids should be maintained in facilities with background colors and light intensities similar to those of the testing area.
(iii) Care and handling. (A) Daphnids should be cultured in dilution water under similar environmental conditions to those used in the test. Organisms should be handled as little as possible. When handling is necessary it should be done as gently, carefully, and quickly as possible. During culturing and acclimation, daphnids should be observed carefully for ephippia and other signs of stress, physical damage and mortality. Dead and abnormal individuals shall be discarded. Organisms that touch dry surfaces or are dropped or injured in handling shall be discarded.
(B) Smooth glass tubes (I.D. greater than 5 mm) equipped with rubber bulb should be used for transferring daphnids with minimal culture media carry-over. Care should be exercised to introduce the daphnids below the surface of any solution to avoid trapping air under the carapace.
(iv) Feeding. A variety of foods (e.g., unicellular green algae) have been demonstrated to be adequate for daphnid culture. Daphnids shall not be fed during testing.
(2) Facilities – (i) Apparatus. (A) Facilities needed to perform this test include: (1) Containers for culturing and acclimating daphnids; (2) a mechanism for controlling and maintaining the water temperature during the culturing, acclimation, and test periods; (3) apparatus for straining particulate matter, removing gas bubbles, or aerating the water as necessary; and (4) an apparatus for providing a 16-hour light and 8-hour dark photoperiod with a 15 to 30 minute transition period. In addition, the flow-through system shall contain appropriate test chambers in which to expose daphnids to the test chemical and an appropriate test substance delivery system.
(B) Facilities should be well ventilated and free of fumes and disturbances that may affect the test organisms.
(C) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions.
(ii) Construction materials. (A) Materials and equipment that contact test solutions should be chosen to minimize sorption of test chemicals from the dilution water and should not contain substances that can be leached into aqueous solution in quantities that can affect the test results.
(B) For static tests, daphnids can be conveniently exposed to the test chemical in 250 ml beakers or other suitable containers.
(C) For flow-through tests, daphnids can be exposed in glass or stainless steel containers with stainless steel or nylon screen bottoms. The containers should be suspended in the test chamber in such a manner to insure that the test solution flows regularly into and out of the container and that the daphnids are always submerged in at least 5 centimeters of test solution. Test chambers can be constructed using 250 ml beakers or other suitable containers equipped with screened overflow holes, standpipes or V-shaped notches.
(iii) Dilution water. (A) Surface or ground water, reconstituted water or dechlorinated tap water are acceptable as dilution water if daphnids will survive in it for the duration of the culturing, acclimation and testing periods without showing signs of stress. The quality of the dilution water should be constant and should meet the following specifications:
| Substance | Maximum concentration |
|---|---|
| Particulate matter | 20 mg/liter. |
| Total organic carbon or | 2 mg/liter. |
| Chemical oxygen demand | 5 mg/liter. |
| Un-ionized ammonia | 1 µg/liter. |
| Residual chlorine | |
| Total organophosphorus pesticides | 50 ng/liter. |
| Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) or | 50 ng/liter. |
| Organic chlorine | 25 ng/liter. |
(B) The above water quality parameters under paragraph (d)(2)(iii)(A) of this section shall be measured at least twice a year or whenever it is suspected that these characteristics may have changed significantly. If dechlorinated tap water is used, daily chlorine analysis shall be performed.
(C) If the diluent water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) shall be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilled water. Glass distilled or carbon-filtered deionized water with a conductivity less than 1 µohm/cm is acceptable as the diluent for making reconstituted water.
(iv) Cleaning. All test equipment and test chambers shall be cleaned before each use using standard laboratory procedures.
(v) Test substance delivery system. In flow-through tests, proportional diluters, metering pump systems, or other suitable devices should be used to deliver test chemical to the test chambers. The system shall be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test chemical in each chamber. The general operation of the test substance delivery system should be checked twice during a test. The 24-hour flow through a test chamber shall be equal to at least 5 times the volume of the test chamber. During a test, the flow rates should not vary more than 10 percent from any one test chamber to another.
(3) Test parameters. Environmental parameters of the water contained in test chambers shall be maintained as specified below:
(i) The test temperature shall be 20 °C. Excursions from the test temperature shall be no greater than ±2 °C.
(ii) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test chemical. All treatment and control chambers shall be given the same aeration treatment.
(iii) The number of daphnids placed in a test chamber shall not affect test results. Loading shall not exceed 40 daphnids per liter test solution in the static system. In the flow-through test, loading limits will vary depending on the flow rate of dilution water. Loading shall not cause the dissolved oxygen concentration to fall below the recommended levels.
(iv) Photoperiod of 16 hours light and 8 hours darkness.
(e) Reporting. The sponsor shall submit to the U.S. EPA all data developed by the test that are suggestive or predictive of acute toxicity and all concomitant gross toxicological manifestations. In addition to the reporting requirements prescribed in part 792 – Good Laboratory Practice Standards of this chapter, the reporting of test data shall include the following:
(1) The name of the test, sponsor, testing laboratory, study director, principal investigator, and dates of testing.
(2) A detailed description of the test chemical including its source, lot number, composition (identity and concentration or major ingredients and major impurities), known physical and chemical properties and any carriers or other additives used and their concentrations.
(3) The source of the dilution water, its chemical characteristics (e.g., conductivity, hardness, pH, etc.) and a description of any pretreatment.
(4) Detailed information about the daphnids used as brood stock, including the scientific name and method of verification, age, source, treatments, feeding history, acclimation procedures, and culture method. The age of the daphnids used in the test shall be reported.
(5) A description of the test chambers, the volume of solution in the chambers, the way the test was begun (e.g., conditioning, test chemical additions), the number of test organisms per test chamber, the number of replicates per treatment, the lighting, the method of test chemical introduction or the test substance delivery system and the flow rate (in flow-through test) expressed as volume additions per 24 hours.
(6) The concentration of the test chemical in each test chamber at times designated for static and flow-through tests.
(7) The number and percentage of organisms that were immobilized or showed any adverse effects in each test chamber at each observation period.
(8) Utilizing the average measured test chemical concentration, concentration-response curves should be fitted to immobilization data at 24 and 48 hours. A statistical test of goodness-of-fit should be performed and the results reported.
(9) The 24- and 48-hour EC
(10) All chemical analyses of water quality and test chemical concentrations, including methods, method validations and reagent blanks.
(11) The data records of the culture, acclimation and test temperatures.
(12) Any deviation from this test guideline and anything unusual about the test, e.g., diluter failure, temperature fluctuations, etc.
§ 797.1330 Daphnid chronic toxicity test.
(a) Purpose. This guideline is intended for use in developing data on the chronic toxicity of chemical substances and mixtures (“chemicals”) subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes a chronic toxicity test in which daphnids are exposed to a chemical in a renewal or a flow-through system. The United States Environmental Protection Agency will use data from this test in assessing the hazard a chemical may present to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA), and the definitions in part 792 Good Laboratory Practice Standards of this chapter apply to this test guideline. In addition, the following definitions apply to this guideline:
(1) Brood stock means the animals which are cultured to produce test organisms through reproduction.
(2) Chronic toxicity test means a method used to determine the concentration of a substance in water that produces an adverse effect on a test organism over an extended period of time. In this test guideline, mortality and reproduction (and optionally, growth) are the criteria of toxicity.
(3) EC
(4) Ephippium means a resting egg which develops under the carapace in response to stress conditions in daphnids.
(5) Flow-through means a continuous or intermittent passage of test solution or dilution water through a test chamber or culture tank with no recycling.
(6) Immobilization means the lack of movement by daphnids except for minor activity of the appendages.
(7) Loading means the ratio of daphnid biomass (grams, wet weight) to the volume (liters) of test solution in a test chamber at a point in time or passing through the test chamber during a specific interval.
(8) MATC (Maximum Acceptable Toxicant Concentration) means the maximum concentration at which a chemical can be present and not be toxic to the test organism.
(9) Renewal system means the technique in which test organisms are periodically transferred to fresh test solution of the same composition.
(c) Test procedures – (1) Summary of the test. (i) Test chambers are filled with appropriate volumes of dilution water. In the flow-through test the flow of dilution water through each chamber is then adjusted to the rate desired. The test substance is introduced into each test chamber. The addition of test substance in the flow-through system is done at a rate which is sufficient to establish and maintain the desired concentration of test substance in the test chamber.
(ii) The test is started within 30 minutes after the test substance has been added and uniformly distributed in the test chambers in the renewal test or after the concentration of test substance in each test chamber of the flow-through test system reaches the prescribed level and remains stable. At the initiation of the test, daphnids which have been cultured or acclimated in accordance with the test design, are randomly placed into the test chambers. Daphnids in the test chambers are observed periodically during the test, immobile adults and offspring produced are counted and removed, and the findings are recorded. Dissolved oxygen concentration, pH, temperature, the concentration of test substance, and other water quality parameters are measured at specified intervals in selected test chambers. Data are collected during the test to determine any significant differences (p≤0.05) in immobilization and reproduction as compared to the control.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted to establish test solution concentrations for the definitive test.
(ii) The daphnids should be exposed to a series of widely spaced concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually under static conditions.
(iii) A minimum of five daphnids should be exposed to each concentration of test substance for a period of time which allows estimation of appropriate chronic test concentrations. No replicates are required and nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to determine concentration-response curves, EC
(ii) A minimum of 20 daphnids per concentration shall be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/l). An equal number of daphnids shall be placed in two or more replicates. The concentration ranges shall be selected to determine the concentration-response curves, EC
(iii) Every test shall include controls consisting of the same dilution water, conditions, procedures and daphnids from the same population (culture container), except that none of the chemical is added.
(iv) The test duration is 21 days. The test is unacceptable if:
(A) More than 20 percent of the control organisms appear to be immobilized, stressed or diseased during the test.
(B) Each control daphnid living the full 21 days produces an average of less than 60 young.
(C) Any ephippia are produced by control animals.
(v) The number of immobilized daphnids in each chamber shall be recorded on day 21 of the test. After offspring are produced, they shall be counted and removed from the test chambers every 2 or 3 days. Concentration-response curves, EC
(vi) In addition to immobility, any abnormal behavior or appearance shall also be reported.
(vii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner as in a way in which appropriate statistical analyses can be used to determine the variation due to placement.
(5) [Reserved]
(6) Analytical measurements. (i) Test chemical. Deionized water should be used in making stock solutions of the test substance. Standard analytical methods should be used whenever available in performing the analyses. The analytical method used to measure the amount of test substance in a sample shall be validated before beginning the test by appropriate laboratory practices. An analytical method is not acceptable if likely degradation products of the test substance, such as hydrolysis and oxidation products, give positive or negative interferences which cannot be systematically identified and corrected mathematically.
(ii) Numerical. The number of immobilized adults, total offspring per adult, and immobilized offspring per adult shall be counted during each test. Appropriate statistical analyses should provide a goodness-of-fit determination for the adult immobilization concentration-response curves calculated on day 21. A 21-day EC
(d) Test conditions – (1) Test species – (i) Selection. (A) The cladocerans, Daphnia magna or D. pulex, are the species to be used in this test. Either species can be utilized for testing of a particular chemical. The species identity of the test organisms should be verified using appropriate systematic keys.
(B) First instar daphnids, ≤24 hours old, are to be used to start the test.
(ii) Acquisition. (A) Daphnids to be used in chronic toxicity tests should be cultured at the test facility. Records should be kept regarding the source of the initial stock and culturing techniques. All organisms used for a particular test shall have originated from the same culture population.
(B) Daphnids shall not be used for a test if:
(1) Cultures contain ephippia.
(2) Adults in the cultures do not produce young before day 12.
(3) More than 20 percent of the culture stock die in the 2 days preceding the test.
(4) Adults in the culture do not produce an average of at least 3 young per adult per day over the 7-day period prior to the test.
(5) Daphnids have been used in any portion of a previous test either in a treatment or in a control.
(iii) Feeding. (A) During the test the daphnids shall be fed the same diet and with the same frequency as that used for culturing and acclimation. All treatments and control(s) shall receive, as near as reasonably possible, the same ration of food on a per-animal basis.
(B) The food concentration depends on the type used. Food concentrations should be sufficient to support normal growth and development and to allow for asexual (parthenogenic) reproduction. For automatic feeding devices, a suggested rate is 5 to 7 mg food (either solids or algal cells, dry weight) per liter dilution water or test solution. For manual once-a-day feeding, a suggested rate is 15 mg food (dry weight) per liter dilution water or test solution.
(iv) Loading. The number of test organisms placed in a test chamber shall not affect test results. Loading shall not exceed 40 daphnids per liter in the renewal system. In the flow-through test, loading limits will vary depending on the flow rate of the dilution water. Loading shall not cause the dissolved oxygen concentration to fall below the recommended level.
(v) Care and handling of test organisms. (A) Daphnids should be cultured in dilution water under similar environmental conditions to those used in the test. A variety of foods have been demonstrated to be adequate for daphnid culture. They include algae, yeasts and a variety of mixtures.
(B) Organisms should be handled as little as possible. When handling is necessary it should be done as gently, carefully, and quickly as possible. During culturing and acclimation, daphnids should be observed carefully for ephippia and other signs of stress, physical damage, and mortality. Dead and abnormal individuals shall be discarded. Organisms that touch dry surfaces or are dropped or injured during handling shall be discarded.
(C) Smooth glass tubes (I.D. greater than 5mm) equipped with a rubber bulb can be used for transferring daphnids with minimal culture media carry-over.
(D) Care should be exercised to introduce the daphnids below the surface of any solution so as not to trap air under the carapace.
(vi) Acclimation. (A) Brood daphnids shall be maintained in 100 percent dilution water at the test temperature for at least 48 hours prior to the start of the test. This is easily accomplished by culturing them in dilution water at the test temperature. During acclimation, daphnids shall be fed the same food as will be used for the definitive test.
(B) During culturing and acclimation to the dilution water, daphnids should be maintained in facilities with background colors and light intensities similar to those of the testing area.
(2) Facilities – (i) General. (A) Facilities needed to perform this test include:
(1) Containers for culturing and acclimating daphnids.
(2) A mechanism for controlling and maintaining the water temperature during the culturing, acclimation and test periods.
(3) Apparatus for straining particulate matter, removing gas bubbles, or aerating the water when water supplies contain particulate matter, gas bubbles, or insufficient dissolved oxygen, respectively.
(4) An apparatus for providing a 16-hour light and 8-hour dark photoperiod.
(5) An apparatus to introduce food if continuous or intermittent feeding is used.
(6) In addition, the flow-through test shall contain appropriate test chambers in which to expose daphnids to the test substance and an appropriate test substance delivery system.
(B) Facilities should be well ventilated and free of fumes and other disturbances that may affect the test organisms.
(ii) Test chambers. (A) Materials and equipment that contact test solutions should be chosen to minimize sorption of test chemicals from the dilution water and should not contain substances that can be leached into aqueous solution in quantities that can affect test results.
(B) For renewal tests, daphnids can be conveniently exposed to the test solution in 250 ml beakers or other suitable containers.
(C) For flow-through tests daphnids can be exposed in glass or stainless steel containers with stainless steel or nylon screen bottoms. Such containers shall be suspended in the test chamber in such a manner to ensure that the test solution flows regularly into and out of the container and that the daphnids are always submerged in at least 5 centimeters of test solution. Test chambers can be constructed using 250 ml beakers or other suitable containers equipped with screened overflow holes, standpipes or V-shaped notches.
(D) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions.
(iii) Test substance delivery system. (A) In the flow-through test, proportional diluters, metering pump systems or other suitable systems should be used to deliver the test substance to the test chambers.
(B) The test substance delivery system shall be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test substance in each chamber. The general operation of the test substance delivery system should be checked twice daily during a test. The 24-hour flow rate through a test chamber shall be equal to at least five times the volume of the test chamber. During a test, the flow rates shall not vary more than 10 percent from any one test chamber to another. For the renewal test, test substance dilution water shall be completely replaced at least once every 3 days.
(iv) Dilution water. (A) Surface or ground water, reconstituted water, or dechlorinated tap water are acceptable as dilution water if daphnids will survive in it for the duration of the culturing, acclimation, and testing periods without showing signs of stress. The quality of the dilution water should be constant and should meet the following specificiations:
| Substance | Maximum concentration |
|---|---|
| Particulate matter | 20 mg/l. |
| Total organic carbon or | 2 mg/l. |
| Chemical oxygen demand | 5 mg/l. |
| Un-ionized ammonia | 20 µg/l. |
| Residual chlorine | |
| Total organophosphorus pesticides | 50 ng/l. |
| Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) | 50 ng/l. |
| or organic chlorine | 25 ng/l. |
(B) The water quality characteristics listed above shall be measured at least twice a year or when it is suspected that these characteristics may have changed significantly. If dechlorinated tap water is used, daily chlorine analysis shall be performed.
(C) If the diluent water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) shall be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilled water. Glass distilled or carbon filtered deionized water with a conductivity of less than 1 microohm/cm is acceptable as the diluent for making reconstituted water.
(D) If the test substance is not soluble in water an appropriate carrier should be used.
(v) Cleaning of test system. All test equipment and test chambers shall be cleaned before each use following standard laboratory procedures. Cleaning of test chambers may be necessary during the testing period.
(3) Test parameters. (i) Environmental conditions of the water contained in test chambers should be maintained as specified in this paragraph:
(A) The test temperature shall be 20 °C. Excursions from the test temperature shall be no greater than ±2 °C.
(B) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test substance. All treatment and control chambers shall be given the same aeration treatment.
(C) Photoperiod of 16-hours light and 8-hours darkness.
(ii) Additional measurements include:
(A) The concentration of the test substance in the chambers shall be measured during the test.
(B) At a minimum, the concentration of test substance should be measured as follows:
(1) In each chamber before the test.
(2) In each chamber on days 7, 14, and 21 of the test.
(3) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. Equal aliquots of test solution may be removed from each replicate chamber and pooled for analysis. Among replicate test chambers of a treatment concentration, the measured concentration of the test substance should not vary more than 20 percent.
(4) An apparatus for providing a 16-hour light and 8-hour dark photoperiod.
(C) The dissolved oxygen concentration, temperature and pH shall be measured at the beginning of the test and on days 7, 14, and 21 in at least two chambers of the high, middle, low, and control test concentrations.
(e) Reporting. The sponsor shall submit to the U.S. Environmental Protection Agency all data developed by the test that are suggestive or predictive of chronic toxicity and all associated toxicologic manifestations. In addition to the reporting requirements prescribed in the part 792 – Good Laboratory Practice Standards of this chapter the reporting of test data shall include the following:
(1) The name of the test, sponsor, testing laboratory, study director, principal investigator, and dates of testing.
(2) A detailed description of the test substance including its source, lot number, composition (identity and concentration of major ingredients and major impurities), known physical and chemical properties, and any carriers or other additives used and their concentrations.
(3) The source of the dilution water, its chemical characteristics (e.g., conductivity, hardness, pH), and a description of any pretreatment.
(4) Detailed information about the daphnids used as brood stock, including the scientific name and method of verification, age, source, treatments, feeding history, acclimation procedures, and culture methods. The age of the daphnids used in the test shall be reported.
(5) A description of the test chambers, the volume of solution in the chambers, the way the test was begun (e.g., conditioning, test substance additions), the number of test organisms per test chamber, the number of replicates per treatment, the lighting, the renewal process and schedule for the renewal chronic test, the test substance delivery system and flow rate expressed as volume additions per 24 hours for the flow-through chronic test, and the method of feeding (manual or continuous) and type of food.
(6) The concentration of the test substance in test chambers at times designated for renewal and flow-through tests.
(7) The number and percentage of organisms that show any adverse effect in each test chamber at each observation period.
(8) The cumulative adult and offspring immobilization values and the progeny produced at designated observation times, the time (days) to first brood and the number of offspring per adult in the control replicates and in each treatment replicate.
(9) All chemical analyses of water quality and test substance concentrations, including methods, method validations and reagent blanks.
(10) The data records of the culture, acclimation, and test temperatures.
(11) Any deviation from this test guideline, and anything unusual about the test, (e.g., dilution failure, temperature fluctuations).
(12) The MATC to be reported is calculated as the geometric mean between the lowest measured test substance concentration that had a significant (p≤0.05) effect and the highest measured test substance concentration that had no significant (p≤0.05) effect on day 21 of the test. The most sensitive of the test criteria (number of adult animals immobilized, the number of young per female and the number of immobilized young per female) is used to calculate the MATC. The criterion selected for MATC computation is the one which exhibits an effect (a statistically significant difference between treatment and control groups; p≤0.05) at the lowest test substance concentration for the shortest period of exposure. Appropriate statistical tests (analysis of variance, mean separation test) shall be used to test for significant test substance effects. The statistical tests employed and the results of these tests shall be reported.
(13) Concentration-response curves utilizing the average measured test substance concentration shall be fitted to cumulative adult immobilization data at 21 days. A statistical test of goodness-of-fit shall be performed and the results reported.
(14) An EC
§ 797.1400 Fish acute toxicity test.
(a) Purpose. This guideline may be used to develop data on the acute toxicity of chemical substances and mixtures (“chemicals”) subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes tests to be used to develop data on the acute toxicity of chemicals to fish. The United States Environmental Protection Agency (EPA) will use data from these tests in assessing the hazard of a chemical to the environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA), and the definitions in part 792 – Good Laboratory Practice Standards of this chapter apply to this test guideline. The following definitions also apply to this guideline:
(1) Acclimation means the physiological compensation by test organisms to new environmental conditions (e.g., temperature, hardness, pH).
(2) Acute toxicity test means a method used to determine the concentration of a substance that produces a toxic effect on a specified percentage of test organisms in a short period of time (e.g., 96 hours). In this guideline, death is used as the measure of toxicity.
(3) Carrier means a solvent used to dissolve a test substance prior to delivery to the test chamber.
(4) Conditioning means the exposure of construction materials, test chambers, and testing apparatus to dilution water or to test solutions prior to the start of a test in order to minimize the sorption of the test substance onto the test facilities or the leaching of substances from the test facilities into the dilution water or test solution.
(5) Death means the lack of opercular movement by a test fish.
(6) Flow-through means a continuous or an intermittent passage of test solution or dilution water through a test chamber, or a holding or acclimation tank with no recycling.
(7) Incipient LC
(8) LC
(9) Loading means the ratio of fish biomass (grams, wet weight) to the volume (liters) of test solution in a test chamber or passing through it in a 24-hour period.
(10) Static means the test solution is not renewed during the period of the test.
(11) Test solution means the test substance and the dilution water in which the test substance is dissolved or suspended.
(c) Test procedures – (1) Summary of the test. (i) Test chambers are filled with appropriate volumes of dilution water. If a flow-through test is performed, the flow of dilution water through each chamber is adjusted to the rate desired.
(ii) The test substance is introduced into each test chamber. In a flow-through test, the amount of test substance which is added to the dilution water is adjusted to establish and maintain the desired concentration of test substance in each test chamber.
(iii) Test fish which have been acclimated in accordance with the test design are introduced into the test and control chambers by stratified random assignment.
(iv) Fish in the test and control chambers are observed periodically during the test; dead fish are removed at least twice each day and the findings are recorded.
(v) The dissolved oxygen concentration, pH, temperature and the concentration of test substance are measured at intervals in selected test chambers.
(vi) Concentration-response curves and LC
(2) [Reserved]
(3) Range finding test. If the toxicity of the test substance is not already known, a range finding test should be performed to determine the range of concentrations to be used in the definitive test. The highest concentration of test substance for use in the range finding test should not exceed its solubility in water or the permissible amount of the carrier used.
(4) Definitive test. (i) A minimum of 20 fish should be exposed to each of five or more test substance concentrations. The range of concentrations to which the fish are exposed should be such that in 96 hours there are at least two partial mortality exposures bracketing 50 percent survival.
(ii) For exposure to each concentration of a test substance, an equal number of test fish shall be placed in two or more replicate test chambers. Test fish shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions.
(iii) Every test shall include a control consisting of the same dilution water, conditions, procedures, and fish from the same group used in the test, except that none of the test substance is added.
(iv) Mortality data collected during the test are used to calculate a 96-hour LC
(v) Test fish shall not be fed while they are being exposed to the test substance under static conditions or during the first 96 hours of flow-through testing. If the test continues past 96 hours, the fish should be fed a suitable food at a maintenance level every other day beginning on test day 5. Any excess food and the fecal material should be removed when observed.
(5) Test results. (i) Death is the primary criterion used in this test guideline to evaluate the toxicity of the test substance.
(ii) In addition to death, any abnormal behavior such as, but not limited to, erratic swimming, loss of reflex, increased excitability, lethargy, or any changes in appearance or physiology such as discoloration, excessive mucous production, hyperventilation, opaque eyes, curved spine, or hemorrhaging shall be recorded.
(iii) Observations on compound solubility shall be recorded. The investigator shall report the appearance of surface slicks, precipitates, or material adhering to the sides of the test chamber.
(iv) Each test and control chamber shall be checked for dead fish and observations recorded at 24, 48, 72, and 96 hours after the beginning of the test or within one hour of the designated times. If the test is continued past 96 hours, additional observations shall be made every 24 hours until termination.
(v) The mortality data is used to calculate LC
(vi) A test is unacceptable if more than 10 percent of the control fish die or exhibit abnormal behavior during a 96-hour test. If a flow-through test is continued past 96 hours, the maximum allowable additional mortality is 10 percent.
(6) Analytical measurements – (i) Water quality analysis. (A) The hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and particulate matter of the dilution water should be measured at the beginning of each static test and at the beginning and end of each flow-through test. The month to month variation of the above values should be less than 10 percent and the pH should vary less than 0.4 units.
(B) During static tests, the dissolved oxygen concentration, temperature, and pH shall be measured in each test chamber at the beginning and end of the test. The test solution volume shall not be reduced by more than 10 percent as a result of these measurements.
(C) During flow-through tests, dissolved oxygen, temperature and pH measurements shall be made in each chamber at the beginning and end of the test.
(ii) Collection of samples for measurement of test substance. Test solution samples to be analyzed for the test substance should be taken midway between the top, bottom, and sides of the test chamber. These samples should not include any surface scum or material dislodged from the bottom or sides. Samples should be analyzed immediately or handled and stored in a manner which minimizes loss of test substance through microbial degradation, photodegradation, chemical reaction, volatilization, or sorption.
(iii) Measurement of test substance. (A) For static tests, the concentration of the test substance shall be measured at a minimum in each test chamber at each test concentration at the beginning (0-hour, before fish are added) and at the end of the test. During flow-through tests, the concentration of test substance shall be measured as follows:
(1) In at least the chamber of each test concentration at 0-hour.
(2) In at least the chamber of each test concentration at 96-hours and every 4 days thereafter, as long as the test is continued.
(3) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system.
(4) Equal aliquots of test solution may be removed from each replicate chamber and pooled for analysis.
(B) Filters and their holders used for determining the dissolved test substance concentrations should be prewashed with several volumes of distilled water and undergo a final rinse with test solution. Glass or stainless steel filter holders are best for organic test substances, while plastic holders are best for metals. The sample should be filtered within 30 minutes after it is taken from the test chamber.
(C) The analytical methods used to measure the amount of test substance in a sample shall be validated before beginning the test. The accuracy of a method should be verified by a method such as using known additions. This involves adding a known amount of the test substance to three water samples taken from a chamber containing dilution water and the same number and species of fish as are used in the test. The nominal concentration of the test substance in those samples should span the concentration range to be used in the test.
(D) An analytical method is not acceptable if likely degradation products of the test substance give positive or negative interferences, unless it is shown that such degradation products are not present in the test chambers during the test.
(E) In addition to analyzing samples of test solution, at least one reagent blank, containing all reagents used, should also be analyzed.
(F) If the measured concentrations of dissolved test substance are considerably lower (e.g.,
(G) Among replicate test chambers, the measured concentrations shall not vary more than 20 percent. The measured concentration of the test substance in any chamber during the test should not vary more than 30 percent from the measured concentration at time 0.
(H) The mean measured concentration of test substance shall be used to calculate all LC
(d) Test conditions – (1) Test species – (i) Selection. The test species for this test are the rainbow trout (Salmo gairdneri), bluegill (Lepomis macrochirus) and fathead minnow (Pimephales promelas). The particular species of fish to be used will be prescribed in the test rule.
(ii) Age and condition of fish. (A) Juvenile fish shall be used. Fish used in a particular test shall be the same age and be of normal size and appearance for their age. The longest fish shall not be more than twice the length of the shortest.
(B) All newly acquired fish should be quarantined and observed for at least 14 days prior to use in a test.
(C) Fish shall not be used for a test if they appear stressed or if more than five percent die during the 48 hours immediately prior to the test.
(iii) Acclimation of test fish. (A) If the holding water is not from the same source as the test dilution water, acclimation to the dilution water should be done gradually over a 48-hour period. The fish should then be held an additional 14 days in the dilution water prior to testing. Any changes in water temperature should not exceed 3 °C per day. Fish should be held for a minimum of 7 days at the test temperature prior to testing.
(B) During the final 48-hours of acclimation, fish should be maintained in facilities with background colors and light intensities similar to those of the testing area and should not be fed.
(2) Facilities – (i) General. Facilities needed to perform this test include:
(A) Flow-through tanks for holding and acclimating fish.
(B) A mechanism for controlling and maintaining the water temperature during the holding, acclimation and test periods.
(C) Apparatus for straining particulate matter, removing gas bubbles, or insufficient dissolved oxygen, respectively.
(D) Apparatus for providing a 16-hour light and 8-hour dark photoperiod with a 15- to 30-minute transition period.
(E) Chambers for exposing test fish to the test substance.
(F) A test substance delivery system for flow-through tests.
(ii) Construction materials. Construction materials and commercially purchased equipment that may contact the stock solution, test solution, or dilution water should not contain substances that can be leached or dissolved into aqueous solutions in quantities that can alter the test results. Materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test chemicals. Glass, stainless steel, and perfluorocarbon plastic should be used whenever possible. Concrete, fiberglass, or plastic (e.g., PVC) may be used for holding tanks, acclimation tanks, and water supply systems, but they should be used to remove rust particles. Rubber, copper, brass, galvanized metal, epoxy glues, and lead should not come in contact with the dilution water, stock solution, or test solution.
(iii) Test substance delivery system. In flow-through tests, diluters, metering pump systems, or other suitable devices should be used to deliver the test substance to the test chambers. The system used should be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test substance delivered to each chamber. The general operation of the test substance delivery system should be checked twice daily during a test. The 24-hour flow rate through a test chamber should be a minimum of 6 tank volumes. During a test, the flow rates should not vary more than 10 percent from one test chamber to another.
(iv) Test chambers. Test chambers made of stainless steel should be welded, not soldered. Test chambers made of glass should be fused or bonded using clear silicone adhesive. As little adhesive as possible should be left exposed in the interior of the chamber.
(v) Cleaning of test system. Test substance delivery systems and test chambers should be cleaned before each test. They should be washed with detergent and then rinsed in sequence with clean water, pesticide-free acetone, clean water, and 5 percent nitric acid, followed by two or more changes of dilution water.
(vi) Dilution water. (A) Clean surface or ground water reconstituted water, or dechlorinated tap water is acceptable as dilution water if the test fish will survive in it for the duration of the holding, acclimating, and testing periods without showing signs of stress, such as discoloration, hemorrhaging, disorientation or other unusual behavior. The quality of the dilution water should be constant and should meet the following specifications measured at least twice a year:
| Substance | Maximum |
|---|---|
| Particulate matter | 20 mg/liter. |
| Total organic carbon or | 2 mg/liter. |
| chemical oxygen demand | 5 mg/liter. |
| Un-ionized ammonia | 1 µg/liter. |
| Residual chlorine | 1 µg/liter. |
| Total organochloring pesticides | 50 µg/liter. |
| Total organocholorine pesticides plus polychlorinated biphenyls (PCBs) | 50 µg/liter. |
| or organic chlorine | 25 µg/liter. |
(B) The concentration of dissolved oxygen in the dilution water should be between 90 and 100 percent saturation; 9.8 to 10.9 mg/l for tests with trout, and 8.0 to 8.9 mg/l for tests with bluegill or fathead minnow at sea level. If necessary, the dilution water can be aerated before the addition of the test substance. All reconstituted water should be aerated before use. Buffered soft water should be aerated before but not after the addition of buffers.
(C) If disease organisms are present in the dilution water in sufficient numbers to cause infection, they should be killed or removed by suitable equipment.
(D) Glass distilled or carbon filtered deionized water with a conductivity less than 1 micromho/cm is acceptable for use in making reconstituted water. If the reconstituted water is prepared from a ground or surface water source, conductivity, and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured on each batch.
(vii) Carriers. (A) Distilled water should be used in making stock solutions of the test substance. If the stock volume however is more than 10 percent of the test solution volume, dilution water should be used. If a carrier is absolutely necessary to dissolve the test substance, the volume used should not exceed the minimum volume necessary to dissolve or suspend the test substance in the test solution. If the test substance is a mixture, formulation, or commercial product, none of the ingredients is considered a carrier unless an extra amount is used to prepare the stock solution.
(B) Triethylene glycol and dimethyl formamide are the preferred carriers, but acetone may also be used. The concentration of triethylene glycol in the test solution should not exceed 80 mg/1. The concentration of dimethyl formamide or acetone in the test solution should not exceed 5.0 mg/1.
(3) Test parameters – (i) Loading. The number of fish placed in a test chamber should not be so great as to affect the results of the test. The loading should not be so great that the test substance concentrations are decreased by more than 20 percent due to uptake by the fish. In static tests, loading should not exceed 0.5 grams of fish per liter of solution in the test chamber at any one time. In flow-through tests loading should not exceed 0.5 grams of fish per liter of test solution passing through the chamber in 24 hours. These loading rates should be sufficient to maintain the dissolved oxygen concentration above the recommended levels and the ammonia concentration below 20 µg/l.
(ii) Dissolved oxygen concentration. (A) During static tests with rainbow trout the dissolved oxygen in each test chamber shall be greater than 5.5 mg/1. In tests with bluegill and fathead minnows, the DO shall be maintained above 4.5 mg/1.
(B) During flow-through tests the dissolved oxygen concentration shall be maintained above 8.2 mg/1 in tests with trout and above 6.6 mg/l in tests with bluegills or fathead minnows.
(iii) Temperature. The test temperature shall be 22 °C for bluegill and fathead minnow and 12 °C for rainbow trout. Excursions from the test temperature shall be no greater than ±2 °C. The temperature shall be measured at least hourly in one test chamber.
(iv) Light. A 16-hour light and 8-hour dark photoperiod should be maintained.
(e) Reporting. The sponsor shall submit to the EPA all data developed by the test that are suggestive or predictive of toxicity. In addition to the reporting requirements prescribed in part 792 – Good Laboratory Practice Standards of this chapter, the reported test data shall include the following:
(1) The source of the dilution water, a description of any pretreatment, and the measured hardness, acidity, alkalinity, pH, conductivity, TOC or COD and particulate matter.
(2) A description of the test chambers, the depth and volume of solution in the chamber, the specific way the test was begun (e.g., conditioning, test substance additions), and for flow-through tests, a description of the test substance delivery system.
(3) Detailed information about the test fish, including the scientific name and method of verification, average weight (grams, wet weight), standard length, age, source, history, observed diseases, treatments, and mortalities, acclimation procedures, and food used.
(4) The number of replicates used, the number of organisms per replicate, the loading rate, and the flow rate for flow-through tests.
(5) The measured DO, pH and temperature and the lighting regime.
(6) The solvent used, the test substance concentration in the stock solution, the highest solvent concentration in the test solution and a description of the solubility determinations in water and solvents if used.
(7) The concentrations of the test substance at each test concentration just before the start of the test and at all subsequent sampling periods.
(8) The number of dead and live tests organisms, the percentage of organisms that died, and the number that showed any abnormal effects in the control and in each test chamber at each observation period.
(9) The 96-hour LC
(10) When observed, the observed no effect concentration (the highest concentration tested at which there were no mortalities or abnormal behavioral or physiological effects).
(11) The concentration-response curve at each observation period for which a LC
(12) Methods and data records of all chemical analyses of water quality parameters and test substance concentrations, including method validations and reagent blanks.
§ 797.1600 Fish early life stage toxicity test.
(a) Purpose. This guideline is intended to be used for assessing the propensity of chemical substances to produce adverse effects to fish during the early stages of their growth and development. This guideline describes the conditions and procedures for the continuous exposure of several representative species to a chemical substance during egg, fry and early juvenile life stages. The Environmental Protection Agency (EPA) will use data from this test in assessing the potential hazard of the test substance to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA) and the definitions in part 792 – Good Laboratory Practice Standards, apply to this section. In addition, the following definitions are applicable to this specific test guideline:
(1) “Acclimation” physiological or behavioral adaptation of organisms to one or more environmental conditions associated with the test method (e.g., temperature, hardness, pH).
(2) “Carrier” solvent or other agent used to dissolve or improve the solubility of the test substance in dilution water.
(3) “Conditioning” exposure of construction materials, test chambers, and testing apparatus to dilution water or to the test solution prior to the start of the test in order to minimize the sorption of test substance onto the test facilities or the leachig of substances from test facilities into the dilution water or the test solution.
(4) “Control” an exposure of test organisms to dilution water only or dilution water containing the test solvent or carrier (no toxic agent is intentionally or inadvertently added).
(5) “Dilution water” the water used to produce the flow-through conditions of the test to which the test substance is added and to which the test species is exposed.
(6) “Early life stage toxicity test” a test to determine the minimum concentration of a substance which produces a statistically significant observable effect on hatching, survival, development and/or growth of a fish species continuously exposed during the period of their early development.
(7) “Embryo cup” a small glass jar or similar container with a screened bottom in which the embryos of some species (i.e., minnow) are placed during the incubation period and which is normally oscillated to ensure a flow of water through the cup.
(8) “Flow through” refers to the continuous or very frequent passage of fresh test solution through a test chamber with no recycling.
(9) “Hardness” the total concentration of the calcium and magnesium ions in water expressed as calcium carbonate (mg CaCO
(10) “Loading” the ratio of biomass (grams of fish, wet weight) to the volume (liters) of test solution passing through the test chamber during a specific interval (normally a 24-hr. period).
(11) “No observed effect concentration (NOEC)” the highest tested concentration in an acceptable early life stage test: (i) which did not cause the occurrence of any specified adverse effect (statistically different from the control at the 95 percent level); and (ii) below which no tested concentration caused such an occurrence.
(12) “Observed effect concentration (OEC)” the lowest tested concentration in an acceptable early life stage test: (i) Which caused the occurrence of any specified adverse effect (statistically different from the control at the 95 percent level); and (ii) above which all tested concentrations caused such an occurrence.
(13) “Replicate” two or more duplicate tests, samples, organisms, concentrations, or exposure chambers.
(14) “Stock solution” the source of the test solution prepared by dissolving the test substance in dilution water or a carrier which is then added to dilution water at a specified, selected concentration by means of the test substance delivery system.
(15) “Test chamber” the individual containers in which test organisms are maintained during exposure to test solution.
(16) “Test solution” dilution water with a test substance dissolved or suspended in it.
(17) “Test substance” the specific form of a chemical substance or mixture that is used to develop data.
(c) Test Procedures – (1) Summary of test. (i) The early life stage toxicity test with fish involves exposure of newly fertilized embryos to various concentrations of a test substance. Exposure continues for 28 days post hatch for the minnows and 60 days post hatch for the trout species. During this time various observations and measurements are made in a specific manner and schedule in order to determine the lowest effect and highest no-effect concentrations of the test substance.
(ii) A minimum of five exposure (treatment) concentrations of a test substance and one control are required to conduct an early life stage toxicity test. The concentration of the test substance in each treatment is usually 50 percent of that in the next higher treatment level.
(iii) For each exposure concentration of the test substance and for each control (i.e., regular control and carrier control is required) there shall be:
(A) At least two replicate test chambers, each containing one or more embryo incubation trays or cups; and there shall be no water connections between the replicate test chambers;
(B) At least 60 embryos divided equally in such a manner that test results show no significant bias from the distributions, between the embryo incubation trays or cups for each test concentration and control (i.e., 30 per embryo cup with 2 replicates);
(C) All surviving larvae divided equally between the test chambers for each test concentration and control (e.g., 30 larvae per test chamber with 2 replicates).
(iv) Duration. (A) For fathead minnow and sheepshead minnow a test begins when the newly fertilized minnow embryos (less than 48-hours old) are placed in the embryo cups and are exposed to the test solution concentrations. The test terminates following 28 days of post-hatch exposure, i.e., 28 days after the newly hatched fry are transferred from the embryo cups into the test chambers.
(B) For brook trout and rainbow trout a test begins when newly fertilized trout embryos (less than 96-hours old) are placed in the embryo trays or cups and are exposed to the test solution concentrations. The test terminates following 60 days of post-hatch exposure (for an approximate total exposure period of 90 days).
(C) For silverside a test begins with newly fertilized embryos (less than or equal to 48 hours old) and is terminated 28 days after hatching. The chorionic fibrils should be cut before randomly placing the embryos in the egg incubation cups.
(2) [Reserved]
(3) Range-finding test. (i) A range finding test is normally performed with the test substance to determine the test concentrations to be used in the early life stage toxicity test, especially when the toxicity is unknown. It is recommended that the test substance concentrations be selected based on information gained from a 4- to 10-day flow-through toxicity test with juveniles of the selected test species.
(ii) The highest concentration selected for the early life stage toxicity test should approximate the lowest concentration indicated in any previous testing to cause a significant reduction in survival. The range of concentrations selected is expected to include both observed effect and no-observed effect levels. The dilution factor between concentrations is normally 0.50, however, other dilution factors may be used as necessary.
(4) Definitive test – (i) General. (A) A test shall not be initiated until after the test conditions have been met and the test substance delivery system has been observed functioning properly for 48-hours. This includes temperature stability, flow requirements of dilution water, lighting requirements, and the function of strainers and air traps included in the water-supply system, and other conditions as specified previously.
(B) New holding and test facilities should be tested with sensitive organisms (i.e., juvenile test species or daphnids) before use to assure that the facilities or substances possibly leaching from the equipment will not adversely affect the test organisms during an actual test.
(C) Embryos should be acclimated for as long as practical to the test temperature and dilution water prior to the initiation of the test.
(D) When embryos are received from an outside culture source (i.e., rainbow and brook trout) at a temperature at variance with the recommended test temperature they shall be acclimated to the test temperature. When eggs are received, they should be immediately unpacked and the temperature of the surrounding water determined. Sudden temperature changes should be avoided. Acclimation to the appropriate test temperature should be accomplished within a period of 6 hours, and should incorporate the use of dilution water.
(E) Embryos should be visually inspected prior to placement in the embryo cups or screen trays. All dead embryos shall be discarded. Dead embryos can be discerned by a change in coloration from that of living embryos (e.g., trout embryos turn white when dead). During visual inspection, empty shells, opaque embryos, and embryos with fungus or partial shells attached shall be removed and discarded. If less than 50 percent of the eggs to be used appear to be healthy, all embryos in such a lot shall be discarded.
(ii) Embryo incubation procedures. (A) Embryos can be distributed to the embryo cups or screen trays using a pipette with a large bore or a similar apparatus. Newly-hatched silverside fry are very sensitive to handling; the egg incubation cups should not be handled at all the first 5 days after hatching begins. Just before hatching is expected to begin, the embryos should be transferred to clean incubation cups. Trout embryos can be distributed by using a small container which has been precalibrated to determine the approximate number of embryos it can hold; embryos are measured volumetrically in this manner, and are then poured onto the screen tray (or embryo cup). Trout embryos should be separated on the screen tray so that they are not in contact with each other. A final count will ensure the actual number on the screen tray. After random assignment, the screen trays or embryo cups are placed in the test chambers.
(B) Each day until hatch the embryos are visually examined. Minnow embryos may be examined with the aid of a magnifying viewer. Trout embryos should not be touched. Trout embryos should be maintained in low intensity light or in darkness until 1-week post hatch, and are usually examined with the aid of a flashlight or under low intensity light. Dead embryos should be removed and discarded. Any embryos which are heavily infected with fungus shall be discarded and shall be subtracted from the initial number of embryos used as a basis for the calculations of percentage hatch.
(C) When embryos begin to hatch they should not be handled.
(iii) Initiation of fry exposure. (A) Forty-eight hours after the first hatch in each treatment level, or when hatching is completed, the live young fish shall be counted and transferred from each embryo cup into the appropriate test chamber. For silverside, all surviving fry are not counted until six days after hatching and are not transferred to embryo cups. All of the normal and abnormal fry shall be gently released into the test chamber by allowing the fry to swim out of each embryo cup; nets shall not be used. The trout embryos incubated on screen trays will hatch out in the test chambers, therefore handling of fish is not necessary.
(B) If necessary, fry can be transferred from one replicate embryo cup to the other replicate within a test concentration to achieve equal numbers in each replicate chamber.
(C) The number of live fry, live normal fry, live embryos, dead embryos and unaccounted for embryos for each cup shall be recorded when hatching is deemed complete. Those fry which are visibly (without the use of a dissecting scope or magnifying viewer) lethargic or grossly abnormal (either in swimming behavior or physical appearance) shall be counted. Late hatching embryos shall be left in the embryo cups to determine if they will eventually hatch or not. The range of time-to-hatch (to the nearest day) for each cup shall be recorded.
(iv) Time to first feeding. (A) The first feeding for the fathead and sheepshead minnow fry shall begin shortly after transfer of the fry from the embryo cups to the test chambers. Silversides are fed the first day after hatch. Trout species initiate feeding at swim-up. The trout fry shall be fed trout starter mash three times a day ad libitum, with excess food siphoned off daily. The minnow fry shall be fed live newly-hatched brine shrimp nauplii (Artemia salina) at least three times a day.
(B) For the first seven days, feeding shall be done at minimum intervals of four hours (i.e., 8 am, 12 noon, and 4 pm); thereafter the fry shall be fed as indicated below.
(v) Feeding. (A) The fathead and sheepshead minnow fry shall be fed newly hatched brine shrimp nauplii for the duration of the test at approximately 4-hour intervals three times a day during the week and twice on the weekend after the first week. Trout fry shall be fed at similar intervals and may receive live brine shrimp nauplii in addition to the trout starter food after the first week. Between days 1 and 8 after first hatching, silverside fry are fed the rotifer, Brachionus plicatilis, three times daily at a concentration of 5,000 to 10,000 organisms per egg cup (based on 15 fish/cup). From days 9 to 11, the fry shall be fed approximately 2,500 newly hatched brine shrimp (Artemia) nauplii and 5,000 to 10,000 rotifers twice daily. For the remainder of the test, the fish will be fed brine shrimp exclusively. The number of organisms used should be gradually increased to approximately 5,000 nauplii by test day 28.
(B) An identical amount of food should be provided to each chamber. Fish should be fed ad libitum for 30 minutes with excess food siphoned off the bottom once daily if necessary.
(C) Fish should not be fed for the last 24 hours prior to termination of the test.
(vi) Carriers. Water should be used in making up the test stock solutions. If carriers other than water are absolutely necessary, the amount used should be the minimum necessary to achieve solution of the test substance. Triethylene glycol and dimethyl formamide are preferred, but ethanol and acetone can be used if necessary. Carrier concentrations selected should be kept constant at all treatment levels.
(vii) Controls. Every test requires a control that consists of the same dilution water, conditions, procedures, and test organisms from the same group used in the other test chambers, except that none of the test substance is added. If a carrier (solvent) is used, a separate carrier control is required in addition to the regular control. The carrier control shall be identical to the regular control except that the highest amount of carrier present in any treatment is added to this control. If the test substance is a mixture, formulation, or commercial product, none of the ingredients is considered a carrier unless an extra amount is used to prepare the stock solution.
(viii) Randomization. The location of all test chambers within the test system shall be randomized. A representative sample of the test embryos should be impartially distributed by adding to each cup or screen tray no more than 20 percent of the number of embryos to be placed in each cup or screen tray and repeating the process until each cup or screen tray contains the specified number of embryos. Alternatively, the embryos can be assigned by random assignment of a small group (e.g., 1 to 5) of embryos to each embryo cup or screen tray, followed by random assignment of a second group of equal number to each cup or tray, which is continued until the appropriate number of embryos are contained in each embryo cup or screen tray. The method of randomization used shall be reported.
(ix) Observations. During the embryo exposure period observations shall be made to check for mortality. During the exposure period of the fry, observations shall be made to check for mortality and to note the physical appearance and behavior of the young fish. The biological responses are used in combination with physical and chemical data in evaluating the overall lethal and sublethal effects of the test substance. Additional information on the specific methodology for the data obtained during the test procedure are discussed in the following sections.
(x) Biological data. (A) Death of embryos shall be recorded daily.
(B) When hatching commences, daily records of the number of embryos remaining in each embryo cup are required. This information is necessary to quantify the hatching success. A record of all deformed larvae shall be kept throughout the entire post-hatch exposure. Time to swim-up shall be recorded for the trout. Upon transfer of fry from the embryo cups to the test chambers, daily counts of the number of live fish should be made. At a minimum, live fish shall be counted on days 4, 11, 18, 25 and (weekly thereafter for the trout species) finally on termination of the test.
(C) The criteria for death of young fish is usually immobility, especially absence of respiratory movement, and lack of reaction to gentle prodding. Deaths should be recorded daily and dead fish removed when discovered.
(D) Daily and at termination of the test, the number of fish that appear (without the use of a magnifying viewer) to be abnormal in behavior (e.g., swimming erratic or uncoordinated, obviously lethargic, hyperventilating, or over excited, etc.) or in physical appearance (e.g., hemorrhaging, producing excessive mucous, or are discolored, deformed, etc.) shall be recorded and reported in detail.
(E) All physical abnormalities (e.g., stunted bodies, scoliosis, etc.) shall be photographed and the deformed fish which die, or are sacrificed at the termination of the test, shall be preserved for possible future pathological examination.
(F) At termination, all surviving fish shall be measured for growth. Standard length measurements should be made directly with a caliper, but may be measured photographically. Measurements shall be made to the nearest millimeter (0.1 mm is desirable). Weight measurements shall also be made for each fish alive at termination (wet, blotted dry, and to the nearest 0.01 g for the minnows and 0.1 g for the trout). If the fish exposed to the toxicant appear to be edematous compared to control fish, determination of dry, rather than wet, weight is recommended.
(G) Special physiological, biochemical and histological investigations on embryos, fry, and juveniles may be deemed appropriate and shall be performed on a case by case basis.
(5) Test results. (i) Data from toxicity tests are usually either continuous (e.g. length or weight measurements) or dichotomous (e.g. number hatching or surviving) in nature. Several methods are available and acceptable for statistical analysis of data derived from early life stage toxicity tests; however, the actual statistical methodology to analyze and interpret the test results shall be reported in detail.
(ii) The significance level for all statistical testing shall be a minimum of P = 0.05 (95 percent confidence level).
(A) Example of statistical analysis. (1) Mortality data for the embryonic stage, fry stage and for both stages in replicate exposure chambers should first be analyzed using a two-way analysis of variance (ANOVA) with interaction model. This analysis will determine if replicates are significantly different from each other. If a significant difference between replicates or a significant interaction exists, cause for the difference should be determined. Modification should then be made in the test apparatus or in handling procedures for future toxicity tests. Further calculations should incorporate the separation of replicates. If no significant difference is observed, replicates may be pooled in further analyses.
(2) After consideration of replicate responses, mortality data should then be subjected to one-way ANOVA. The purpose of this analysis is to determine if a significant difference exists in the percentage mortality between control fish and those exposed to the test material.
(3) If the one-way ANOVA results in a F ratio that is significant, it would be acceptable to perform t-tests on the control versus each concentration. A second technique is to identify treatment means that are significantly different; this method should involve the additional assumption that the true mean response decreases generally with increasing concentration. The researcher may also be interested in determining significant differences between concentrations.
(4) Growth data should also be analyzed by one-way ANOVA with the inclusion of a covariate to account for possible differences in growth of surviving fry in embryo cup(s) that contain fewer individuals. This condition can occur in cases when the same amount of food is given to each test chamber regardless of the number of survivors.
(B) Test data to be analyzed. Data to be statistically analyzed are:
(1) Percentage of healthy, fertile embryos at 40-48 hours after initiation of the test. Percentage is based upon initial number used.
(2) Percentage of embryos that produce live fry for release into test chambers. Percentage is based on number of embryos remaining after thinning.
(3) Percentage of embryos that produce live, normal fry for release into test chambers. Percentage is based upon number of embryos remaining after thinning.
(4) Percentage of fry survival at swim-up for trout. Percentage is based upon number of embryos remaining after thinning.
(5) Percentage of embryos that produce live fish at end of test. Percentage is based upon number of embryos remaining after thinning.
(6) Percentage of embryos that produce live, normal fish at end of test. Percentage is based upon number of embryos remaining after thinning.
(7) Weights and lengths of individual fish alive at the end of the test.
(C) It is important that fish length and weight measurements be associated with individual test chambers since the density of the fish and available food should be considered in the growth of the organism.
(iii) Acceptability criteria. (A) An early life stage toxicity test is not acceptable unless at least one of the following criteria is significantly different (p = 0.05) from control organisms when compared with treated organisms, and the responses are concentration-dependent: mortality of embryos, hatching success, mortality of fry (at swim-up for trout), total mortality throughout the test, and growth (i.e. weight). If no significant effects occur, but the concentrations tested were the highest possible due to solubility or other physio-chemical limitations, the data will be considered for acceptance.
(B) In addition to obtaining significant effects on the exposed test species, a measure of acceptability in the response of control fish is also required.
(C) A test is not acceptable if the average survival of the control fish at the end of the test is less than 80 percent or if survival in any one control chamber is less than 70 percent. For silversides, a test is not acceptable if the average overall survival of the control embryos and fish at the end of the test is less than 60 percent.
(D) If a carrier is used, the criteria for effect (mortality of embryos and fry, growth, etc.) used in the comparison of control and exposed test organisms shall also be applied to the control and control with carrier chambers. For the test to be considered acceptable, no significant difference shall exist between these criteria.
(E) A test is not acceptable if the relative standard deviation (RSD = 100 times the standard deviation divided by the mean) of the weights of the fish that were alive at the end of the test in any control test chamber is greater than 40 percent.
(6) Analytical measurements – (i) Analysis of water quality. Measurement of certain dilution water quality parameters shall be performed every 6 months, to determine the consistency of the dilution water quality. In addition, if data in 30-day increments are not available to show that freshwater dilution water is constant, measurements of hardness, alkalinity, pH, acidity, conductivity, TOC or COD and particulate matter should be conducted once a week in the highest test substance concentration. Measurement of calcium, magnesium, sodium, potassium, chloride, and sulfate is desirable.
(ii) Dissolved oxygen measurement. The dissolved oxygen concentration shall be measured in each test chamber at the beginning of the test and at least once weekly thereafter (as long as live organisms are present) in two replicates of the control and the high, medium, and low test substance concentrations.
(iii) Temperature measurement. Temperatures shall be recorded in all test chambers at the beginning of the test, once weekly thereafter and at least hourly in one test chamber. When possible, the hourly measurement shall be alternated between test chambers and between replicates.
(iv) Test substance measurement. (A) Prior to the addition of the test substance to the dilution water, it is recommended that the test substance stock solution be analyzed to verify the concentration. After addition of the test substance, the concentration of test substance should be measured at the beginning of the test in each test concentration and control(s), and at least once a week thereafter. Equal aliquots of test solution may be removed from each replicate chamber and pooled for analysis. If a malfunction in the delivery system is discovered, water samples shall be taken from the affected test chambers immediately and analyzed.
(B) The measured concentration of test substance in any chamber should be no more than 30 percent higher or lower than the concentration calculated from the composition of the stock solution and the calibration of the test substance delivery system. If the difference is more than 30 percent, the concentration of test substance in the solution flowing into the exposure chamber (influent) should be analyzed. These results will indicate whether the problem is in the stock solution, the test substance delivery system or in the test chamber. Measurement of degradation products of the test substance is recommended if a reduction of the test substance concentration occurs in the test chamber.
(v) Sampling and analysis methodology. (A) Generally, total test substance measurements are sufficient; however, the chemical characteristics of the test substance may require both dissolved and suspended test substance measurements.
(B) For measurement of the test substance, water samples shall be taken midway between the top, bottom, and sides of the test chamber and should not include any surface scum or material stirred up from the bottom or sides. Samples of test solutions shall be handled and stored appropriately to minimize loss of test substance by microbial degradation, photodegradation, chemical reaction, volatilization, or sorption.
(C) Chemical and physical analyses shall be performed using standardized methods whenever possible. The analytical method used to measure the concentration of the test substance in the test solution shall be validated before the beginning of the test. At a minimum, a measure of the accuracy of the method should be obtained on each of two separate days by using the method of known additions, and using dilution water from a tank containing test organisms. Three samples should be analyzed at the next-to-lowest test substance concentration. It is also desirable to study the accuracy and precision of the analytical method for test guideline determination by use of reference (split) samples, or interlaboratory studies, and by comparison with alternative, reference, or corroborative methods of analysis.
(D) An analytical method is not acceptable if likely degradation products of the test substance, such as hydrolysis and oxidation products, give positive or negative interferences, unless it is shown that such degradation products are not present in the test chambers during the test. In general, atomic absorption spectrophotometric methods for metals and gas chromatographic methods for organic compounds are preferable to colorimetric methods.
(E) In addition to analyzing samples of test solution, at least one reagent blank also should be analyzed when a reagent is used in the analysis. Also, at least one sample for the method of known additions should be prepared by adding test substance at the concentration used in the toxicity test.
(d) Test conditions – (1) Test species. (i) One or more of the recommended test species will be specified in rules under part 799 of this chapter requiring testing of specific chemicals. The recommended test species are:
(A) Fathead minnow (Pimephales promelas Rafinesque).
(B) Sheepshead minnow (Cyprinodon variegatus).
(C) Brook trout (Salvelinus fontinalis).
(D) Rainbow trout (Salmo gairdneri).
(E) Atlantic silverside (Menidia menidia).
(F) Tidewater silverside (Menidia peninsulae).
(ii) Embryos used to initiate the early life stage test shall be less than 48 hours old for the fathead and sheepshead minnows, silversides, and less than 96 hours old for the brook trout and rainbow trout. In addition, the following requirements shall be met:
(A) All embryos used in the test shall be from the same source. Embryos shall be obtained from a stock cultured in-house when possible, and maintained under the same parameters as specified for the test conditions. When it is necessary to obtain embryos from an external source, caution should be exercised to ensure embryo viability and to minimize the possibility of fungal growth. A description of the brood stock history or embryo source shall be made available to EPA upon request.
(B) Test species shall be cared for and handled properly in order to avoid unnecessary stress. To maintain test species in good condition and to maximize growth, crowding shall be prevented, and the dissolved oxygen level shall be maintained near saturation.
(C) Embryos and fish shall be handled as little as possible. Embryos shall be counted and periodically inspected until hatching begins. When larvae begin to hatch, they shall not be handled. Transfer of minnow larvae from embryo cups to test chambers shall not involve the use of nets. No handling is necessary following introduction into the test chambers until termination of the test.
(D) If fathead minnow embryos are obtained from in-house culture units, the embryos should be gently removed from the spawning substrate. The method for separating the fertilized eggs from the substrate is important and can affect the viability of the embryos; therefore the finger-rolling procedure is recommended.
(E) Disease treatment. Chemical treatments to cure or prevent diseases should not be used before, and should not be used during a test. All prior treatments of brood stock should be reported in detail. Severely diseased organisms should be destroyed.
(2) Test facilities – (i) Construction materials. Construction materials and equipment that contact stock solutions, test solutions, or dilution water into which test embryos or fish are placed should not contain any substances that can be leached or dissolved into aqueous solutions in quantities that can affect test results. Materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test chemicals from dilution water. Glass, #316 stainless steel, nylon screen and perfluorocarbon plastic (e.g., Teflon#) are acceptable materials. Concrete or rigid (unplasticized) plastic may be used for holding and acclimation tanks, and for water supply systems, but they should be thoroughly conditioned before use. If cast iron pipe is used in freshwater supply systems, colloidal iron may leach into the dilution water and strainers should be used to remove rust particles. Natural rubber, copper, brass, galvanized metal, epoxy glues, and flexible tubing should not come in contact with dilution water, stock solutions, or test solutions.
(ii) Test chambers (exposure chambers). (A) Stainless steel test chambers should be welded or glued with silicone adhesive, and not soldered. Glass should be fused or bonded using clear silicone adhesive. Epoxy glues are not recommended, but if used ample curing time should be allowed prior to use. As little adhesive as possible should be in contact with the water.
(B) Many different sizes of test chambers have been used successfully. The size, shape and depth of the test chamber is acceptable if the specified flow rate and loading requirements can be achieved.
(C) The actual arrangement of the test chambers can be important to the statistical analysis of the test data. Test chambers can be arranged totally on one level (tier) side by side, or on two levels with each level having one of the replicate test substance concentrations or controls. Regardless of the arrangement, it shall be reported in detail and considered in the data analysis.
(iii) Embryo incubation apparatus. (A) Recommended embryo incubation apparatus include embryo cups for the minnow species and screen trays for the trout species, although embryo cups can be used for the trout species. Embryo cups are normally constructed from approximately 4-5 cm inside diameter, 7-8 cm high, glass jars with the end cut off or similar sized sections of polyethylene tubing. One end of the jar or tubing is covered with stainless steel or nylon screen (approximately 40 meshes per inch is recommended). Embryo cups for silversides are normally constructed by using silicone adhesive to glue a 10-cm high, 363-um nylon mesh tube inside a 9-cm I.D. glass Petri dish bottom. The embryo cups shall be appropriately labeled and then suspended in the test chamber in such a manner as to ensure that the test solution regularly flows through the cup and that the embryos are always submerged but are not agitated too vigorously. Cups may be oscillated by a rocker arm apparatus with a low rpm motor (e.g., 2 rpm) to maintain the required flow of test water. The vertical-travel distance of the rocker arm apparatus during oscillation is normally 2.5-4.0 cm. The water level in the test chambers may also be varied by means of a self-starting siphon in order to ensure exchange of water in the embryo cups.
(B) The trout embryo incubation trays can be made from stainless steel screen (or other acceptable material such as plastic) of about 3-4 mm mesh. The screen tray should be supported above the bottom of the test chamber by two folds of screen or other devices which function as legs or supports. The edges of the screen tray should be turned up to prevent bump spills and to prevent the embryos from rolling off in the event of excessive turbulence. Suspending or supporting the screen tray off the bottom ensures adequate water circulation around the embryos and avoids contact of embryos with possible bottom debris.
(iv) Test substance delivery system. (A) The choice of a specific delivery system depends upon the specific properties and requirements of the test substance. The apparatus used should accurately and precisely deliver the appropriate amount of stock solution and dilution water to the test chambers. The system selected shall be calibrated before each test. Calibration includes determining the flow rate through each chamber, and the proportion of stock solution to dilution water delivered to each chamber. The general operation of the test substance delivery system shall be checked at least twice daily for normal operation throughout the test. A minimum of five test substance concentrations and one control shall be used for each test.
(B) The proportional diluter and modified proportional diluter systems and metering pump systems have proven suitable and have received extensive use.
(C) Mixing chambers shall be used between the diluter and the test chamber(s). This may be a small container or flow-splitting chamber to promote mixing of test substance stock solution and dilution water, and is positioned between the diluter and the test chambers for each concentration. If a proportional diluter is used, separate delivery tubes shall run from the flow-splitting chamber to each replicate test chamber. Daily checks on this latter system shall be made.
(D) Silverside fry are injured easily and are susceptible to impingement on the mesh of the incubation cups. Consequently, water flow into and out of the cups when counting fry must be at a slow rate. This can be accomplished by using small diameter (e.g., 2 mm I.D.) capillary tubes to drain the test solution from spitter boxes into the replicate test chambers. The use of a self-starting siphon to gradually lower (i.e., less than or equal to 1 min.) the water level approximately 2 cm in the test chamber is recommended. A minimum water depth of 5 cm should be maintained in the cups. Although it may be satisfactory, a rocker-arm type apparatus has not yet been used with silversides.
(v) Other equipment required. (A) An apparatus for removing undesirable organisms, particulate matter and air bubbles.
(B) An apparatus for aerating water.
(C) A suitable magnifying viewer for examination of minnow embryos.
(D) A suitable apparatus for the precise measurement of growth of the fish, including both length (e.g., with metric or ruler caliper or photographic equipment) and weight.
(E) Facilities for providing a continuous supply of live brine shrimp nauplii (Artemia salina).
(F) For silversides, facilities for providing a supply of rotifers (Brachionus plicatilis) for approximately 11 days.
(G) Facilities (or access to facilities) for performing the required water chemistry analyses.
(vi) Cleaning of equipment. (A) Test substance delivery systems and test chambers should be cleaned before use. Test chambers should be cleaned during the test as needed to maintain the dissolved oxygen concentration, and to prevent clogging of the embryo cup screens and narrow flow passages.
(B) Debris can be removed with a rubber bulb and large pipette or by siphoning with a glass tube attached to a flexible hose. Debris should be run into a bucket light enough to observe that no live fish are accidentally discarded.
(vii) Dilution water – (A) General. (1) A constant supply of acceptable dilution water should be available for use throughout the test. Dilution water shall be of a minimum quality such that the test species selected will survive in it for the duration of testing without showing signs of stress (e.g., loss of pigmentation, disorientation, poor response to external stimuli, excessive mucous secretion, lethargy, lack of feeding, or other unusual behavior). A better criterion for an acceptable dilution water for tests on early life stages should be such that the species selected for testing will survive, grow, and reproduce satisfactorily in it.
(2) The concentration of dissolved oxygen in the dilution water (fresh or salt) shall be between 90 percent and 100 percent saturation. When necessary, dilution water should be aerated by means of airstones, surface aerators, or screen tubes before the introduction of the test substance.
(3) Water that is contaminated with undesirable microoganisms (e.g., fish pathogens) shall not be used. If such contamination is suspected, the water should be passed through a properly maintained ultraviolet sterilizer equipped with an intensity meter before use. Efficacy of the sterilizer can be determined by using standard plate count methods.
(B) Freshwater. (1) Natural water (clean surface or ground water) is preferred, however, dechlorinated tap water may be used as a last resort. Reconstituted freshwater is not recommended as a practical dilution water for the early life stage toxicity test because of the large volume of water required.
(2) Particulate and dissolved substance concentrations should be measured at least twice a year and should meet the following specifications:
| Substance | Concentration maximum |
|---|---|
| Particulate matter | |
| Total organic carbon (TOC) | |
| Chemical oxygen demand (COD) | |
| Un-ionized ammonia | |
| Residual chlorine | |
| Total organoposphorus pesticides | |
| Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) | |
| Total organic chlorine |
(3) During any one month, freshwater dilution water should not vary more than 10 percent from the respective monthly averages of hardness, alkalinity and specific conductance; the monthly pH range should be less than 0.4 pH units.
(C) Saltwater. (1) Marine dilution water is considered to be of constant quality if the minimum salinity is greater than 15
(2) Artificial sea salts may be added to natural seawater during periods of low salinity to maintain salinity above 15
(3) Test parameters – (i) Dissolved oxygen concentration. It is recommended that the dissolved oxygen concentration be maintained between 90 and 100 percent saturation; but it shall be no less than 75 percent saturation at all times for both minnow species and between 90 and 100 percent saturation for the trout species in all test chambers. Dilution water in the head box may be aerated, but the test solution itself shall not be aerated.
(ii) Loading and flow rate. (A) The loading in test chambers should not exceed 0.1 grams of fish per liter of test solution passing through the test chamber in 24 hours. The flow rate to each chamber should be a minimum of 6 tank volumes per 24 hours. During a test, the flow rates should not vary more than 10 percent from any one test chamber to any other.
(B) A lower loading or higher flow rate or both shall be used if necessary to meet the following three criteria at all times during the test in each chamber containing live test organisms:
(1) The concentration of dissolved oxygen shall not fall below 75 percent saturation for the fathead and sheepshead minnows and 90 percent for the rainbow and brook trout;
(2) The concentration of un-ionized ammonia should not exceed 1 µg/1; and
(3) The concentration of toxicant should not be lowered (i.e., caused by uptake by the test organisms and/or materials on the sides and bottoms of the chambers) more than 20 percent of the mean measured concentration.
(iii) Temperature. (A) The recommended test temperatures are:
(1) Fathead minnow – 25 °C for all life stages.
(2) Sheepshead minnow – 30 °C for all life stages.
(3) Rainbow and brook trout – 10 °C for embryos. 12 °C for fry and alevins.
(4) Atlantic and tidewater silversides – 25 °C for all life stages.
(B) Excursions from the test temperature shall be no greater than ±2.0 °C. It is recommended that the test system be equipped with an automatic alarm system to alert staff of instantaneous temperature changes in excess of 2 °C. If the water is heated (i.e., for minnow species), precautions should be taken to ensure that supersaturation of dissolved gases is avoided. Temperatures shall be recorded in all test chambers at the beginning of the test and weekly thereafter. The temperature shall be recorded at least hourly in one test chamber throughout the test.
(iv) Light. (A) Brook and rainbow trout embryos shall be maintained in darkness or very low light intensity through one week post-hatch, at which time a 14-hour light and 10-hour dark photoperiod shall be provided.
(B) For fathead and sheepshead minnows, a 16-hour light and 8-hour dark (or 12:12) photoperiod shall be used throughout the test period.
(C) For silversides, a 14-hour light and 10-hour dark photoperiod shall be used throughout the test period.
(D) A 15-minute to 30-minute transition period between light and dark is optional.
(E) Light intensities ranging from 30 to 100 lumens at the water surface shall be provided; the intensity selected should be duplicated as closely as possible for all test chambers.
(e) Reporting. A report of the results of an early life stage toxicity test shall include the following:
(1) Name of test, sponsor, investigator, laboratory, and dates of test duration.
(2) Detailed description of the test substance including its source, lot number, composition (identity and concentration of major ingredients and major impurities), known physical and chemical properties, and any carriers (solvents) or other additives used.
(3) The source of the dilution water, its chemical characteristics, and a description of any pretreatment.
(4) Detailed information about the test organisms including scientific name and how verified and source history, observed diseases, treatments, acclimation procedure, and concentration of any contaminants and the method of measurement.
(5) A description of the experimental design and the test chambers, the depth and volume of the solution in the chambers, the way the test was begun, the number of organisms per treatment, the number of replicates, the loading, the lighting, a description of the test substance delivery system, and the flow rate as volume additions per 24 hours.
(6) Detailed information on feeding of fish during the toxicity test, including type of food used, its source, feeding frequency and results of analysis (i.e., concentrations) for contaminants.
(7) Number of embryos hatched, number of healthy embryos, time to hatch, mortality of embryos and fry, measurements of growth (weight and length), incidence of pathological or histological effects and observations of other effects or clinical signs, number of healthy fish at end of test.
(8) Number of organisms that died or showed an effect in the control and the results of analysis for concentration(s) of any contaminant in the control(s) should mortality occur.
(9) Methods used for, and the results of (with standard deviation), all chemical analyses of water quality and test substance concentration, including validation studies and reagent blanks; the average and range of the test temperature(s).
(10) Anything unusual about the test, any deviation from these procedures, and any other relevant information.
(11) A description of any abnormal effects and the number of fish which were affected during each period between observations in each chamber, and the average concentration of test substance in each test chamber.
(12) Reference to the raw data location.
§ 797.1930 Mysid shrimp acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data on the acute toxicity of chemical substances and mixtures (“chemicals”) subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes a test using mysid shrimp as test organisms to develop data on the acute toxicity of chemicals. The United States Environmental Protection Agency (EPA) will use data from these tests in assessing the hazard of a chemical to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA) and in part 792 – Good Laboratory Practice Standards of this chapter, apply to this test guideline. The following definitions also apply to this guideline.
(1) “Death” means the lack of reaction of a test organism to gentle prodding.
(2) “Flow-through” means a continuous or an intermittent passage of test solution or dilution water through a test chamber or a holding or acclimation tank, with no recycling.
(3) “LC
(4) “Loading” means the ratio of test organisms biomass (grams, wet weight) to the volume (liters) of test solution in a test chamber.
(5) “Retention chamber” means a structure within a flow-through test chamber which confines the test organisms, facilitating observation of test organisms and eliminating loss of organisms in outflow water.
(6) “Static system” means a test chamber in which the test solution is not renewed during the period of the test.
(c) Test procedures – (1) Summary of the test. In preparation for the test, test chambers are filled with appropriate volumes of dilution water. If a flow-through test is performed, the flow of dilution water through each chamber is adjusted to the rate desired. The test substance is introduced into each test chamber. In a flow-through test, the rate at which the test substance is added is adjusted to establish and maintain the desired concentration of test substance in each test chamber. The test is started by randomly introducing mysids acclimated in accordance with the test design into the test chambers. Mysids in the test chambers are observed periodically during the test, the dead mysids removed and the findings recorded. Dissolved oxygen concentration, pH, temperature, salinity, the concentration of test substance, and other water quality characteristics are measured at specified intervals in test chambers. Data collected during the test are used to develop concentration-response curves and LC
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted to determine:
(A) Which life stage (juvenile or young adult) is to be utilized in the definitive test.
(B) The test solution concentrations for the definitive test.
(ii) The mysids should be exposed to a series of widely spaced concentrations of test substance (e.g., 1, 10, 100 mg/l, etc.), usually under static conditions.
(iii) This test should be conducted with both newly hatched juvenile (
(4) Definitive test. (i) The purpose of the definitive test is to determine the concentration-response curves and the 48- and 96-hour LC
(ii) The definitive test shall be conducted on the mysid life stage (juveniles or young adults) which is most sensitive to the test substance being evaluated.
(iii) A minimum of 20 mysids per concentration shall be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). An equal number of mysids shall be placed in two or more replicates. If solvents, solubilizing agents or emulsifiers have to be used, they shall be commonly used carriers and shall not possess a synergistic or antagonistic effect on the toxicity of the test substance. The concentration of solvent shall not exceed 0.1 ml/1. The concentration ranges shall be selected to determine the concentration-response curves and LC
(iv) Every test shall include controls consisting of the same dilution water, conditions, procedures, and mysids from the same population or culture container, except that none of the chemical is added.
(v) The dissolved oxygen concentration temperature, salinity, and pH shall be measured at the beginning and end of the test in each chamber.
(vi) The test duration is 96 hours. The test is unacceptable if more than 10 percent of the control organisms die or exhibit abnormal behavior during the 96 hour test period. Each test chamber should be checked for dead mysids at 24, 48, 72, and 96 hours after the beginning of the test. Concentration-response curves and 24-, 48-, 72- and 96-hour LC
(vii) In addition to death, any abnormal behavior or appearance shall also be reported.
(viii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner or in a way in which appropriated statistical analyses can be used to determine the variation due to placement.
(ix) The concentration of the test substance in the chambers should be measured as often as is feasible during the test. At a minimum, during static tests the concentration of test substance shall be measured at each concentration at the beginning and at the end of the test. During the flow-through test, the concentration of test substance should be measured at the beginning and end of the test and in at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. Equal aliquots of test solution may be removed from each replicate chamber and pooled for analysis. Among replicate test chambers of a treatment concentration, the measured concentration of the test substance should not vary more than 20 percent.
(5) [Reserved]
(6) Analytical measurements – (i) Test chemical. Deionized water should be used in making stock solutions of the test substance. Standard analytical methods should be used whenever available in performing the analyses. The analytical method used to measure the amount of test substance in a sample shall be validated before beginning the test by appropriate laboratory practices. An analytical method is not acceptable if likely degradation products of the test substance, such as hydrolysis and oxidation products, give positive or negative interferences which cannot be systematically identified and corrected mathematically.
(ii) Numerical. The number of dead mysids shall be counted during each definitive test. Appropriate statistical analyses should provide a goodness-of-fit determination for the concentration-response curves. A 48- and 96-hour LC
(d) Test conditions – (1) Test species – (i) Selection. (A) The mysid shrimp, Mysidopsis bahia, is the organism specified for these tests. Either juvenile (
(B) Mysids to be used in chronic toxicity tests should originate from laboratory cultures in order to ensure the individuals are of similar age and experimental history. Mysids used for establishing laboratory cultures may be purchased commercially or collected from appropriate natural areas. Because of similarities with other mysids species, taxonomic verification should be obtained from the commercial supplier by experienced laboratory personnel or by an outside expert.
(C) Mysids used in a particular test shall be of similar age and be of normal size and appearance for their age. Mysids shall not be used for a test if they exhibit abnormal behavior or if they have been used in a previous test, either in a treatment or in a control group.
(ii) Acclimation. (A) Any change in the temperature and chemistry of the dilution water used for holding or culturing the test organisms to those of the test shall be gradual. Within a 24-hour period, changes in water temperature shall not exceed 1 °C, while salinity changes shall not exceed 5 percent.
(B) During acclimation mysids should be maintained in facilities with background colors and light intensities similar to those of the testing areas.
(iii) Care and handling. Methods for the care and handling of mysids such as those described in paragraph (f)(1) of this section can be used during holding, culturing and testing periods.
(iv) Feeding. Mysids should be fed during testing. Any food utilized should support survival, growth and reproduction of the mysids. A recommended food is live Artemia spp. (48-hour-old nauplii).
(2) Facilities – (i) Apparatus. (A) Facilities which may be needed to perform this test include: (1) flow-through or recirculating tanks for holding and acclimating mysids; (2) a mechanism for controlling and maintaining the water temperature during the holding, acclimation and test periods; (3) apparatus for straining particulate matter, removing gas bubbles, or aerating the water, as necessary; and (4) an apparatus for providing a 14-hour light and 10-hour dark photoperiod with a 15 to 30 minute transition period. In addition, for flow-through tests, flow-through chambers and a test substance delivery system are required. Furthermore, it is recommended that mysids be held in retention chambers within test chambers to facilitate observations and eliminate loss of test organisms through outflow water. For static tests, suitable chambers for exposing test mysids to the test substance are required. Facilities should be well ventilated and free of fumes and disturbances that may affect the test organisms.
(B) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions.
(ii) Cleaning. Test substance delivery systems and test chambers shall be cleaned before each test following standard laboratory practices.
(iii) Construction materials. (A) Materials and equipment that contact test solutions should be chosen to minimize sorption of test chemicals from dilution water and should not contain substances that can be leached into aqueous solution in quantities that can affect test results.
(B) For use in the flow-through test, retention chambers utilized for confinement of test organisms can be constructed with netting material of appropriate mesh size.
(iv) Dilution water. (A) Natural or artificial seawater is acceptable as dilution water if mysids will survive and successfully reproduce in it for the duration of the holding, acclimating and testing periods without showing signs of stress, such as reduced growth and fecundity. Mysids shall be cultured and tested in dilution water from the same origin.
(B) Natural seawater shall be filtered through a filter with a pore size of
(C) Artificial seawater can be prepared by adding commercially available formulations or by adding specific amounts of reagent-grade chemicals to deionized water. Deionized water with a conductivity less than 1 µohm/cm at 12 °C is acceptable for making artificial seawater. When deionized water is prepared from a ground or surface water source, conductivity and total organic carbon (or chemical oxygen demand) shall be measured on each batch.
(v) Test substance delivery system. In flow-through tests, proportional diluters, metering pumps, or other suitable systems should be used to deliver test substance to the test chambers. The system used shall be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test substance in each chamber. The general operation of the test substance delivery system should be checked twice daily during a test. The 24-hour flow through a test chamber shall be equal to at least 5 times the volume of the test chamber. During a test, the flow rates should not vary more than 10 percent among test chambers or across time.
(3) Test parameters. Environmental parameters of the water contained in test chambers shall be maintained as specified below:
(i) The test temperature shall be 25 °C. Excursions from the test temperature shall be not greater than ±2 °C.
(ii) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test substance. All treatment and control chambers shall be given the same aeration treatment.
(iii) The number of mysids placed in a test solution shall not be so great as to affect results of the test. Loading shall not exceed 30 mysids per liter for a static test. Loading requirements for the flow-through test will vary depending on the flow rate of dilution water. The loading shall not cause the dissolved oxygen concentration to fall below the recommended levels.
(iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15 to 30 minute transition period.
(v) Salinity of 20 parts per thousand ±3 percent.
(e) Reporting. The sponsor shall submit to the EPA all data developed during the test that are suggestive or predictive of acute toxicity and all concomitant toxicologic manifestations. In addition to the general reporting requirements prescribed in part 792 – Good Laboratory Practice Standards of this chapter, the reporting of test data shall include the following:
(1) The source of the dilution water, its chemical characteristics (e.g., salinity, pH, etc.) and a description of any pretreatment.
(2) Detailed information about the test organisms, including the scientific name and method of verification, age, source, history, abnormal behavior, acclimation procedures and food used.
(3) A description of the test chambers, the depth and volume of solution in the chamber, the way the test was begun (e.g., conditioning, test substance additions, etc.), the number of organisms per treatment, the number of replicates, the loading, the lighting, the test substance delivery system and the flow rate expressed as volume additions per 24 hours.
(4) The measured concentration of test substance in test chambers at the times designated.
(5) The number and percentage of organisms that died or showed any other adverse effects in the control and in each treatment at each observation period.
(6) Concentration-response curves shall be fitted to mortality data collected at 24, 48, 72, and 96 hours. A statistical test of goodness-of-fit shall be performed and the results reported.
(7) The 96-hour LC
(8) Methods and data records of all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks.
(9) The data records of the holding, acclimation and test temperature and salinity.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency, “Bioassay Procedures for the Ocean Disposal Permit Program,” EPA Report No. 600-9-78-010 (Gulf Breeze, Florida, 1978).
(2) [Reserved]
§ 797.1950 Mysid shrimp chronic toxicity test.
(a) Purpose. This guideline is intended for use in developing data on the chronic toxicity of chemical substances and mixtures (“chemicals”) subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes tests using mysids as test organisms to develop data on the chronic toxicity of chemicals. The United States Environmental Protection Agency (EPA) will use data from these tests in assessing the hazard of a chemical to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA) and in part 792 – Good Laboratory Practice Standards of this chapter apply to this test guideline. The following definitions also apply to this guideline:
(1) “Chronic toxicity test” means a method used to determine the concentration of a substance that produces an adverse effect from prolonged exposure of an organism to that substance. In this test, mortality, number of young per female and growth are used as measures of chronic toxicity.
(2) “Death” means the lack of reaction of a test organism to gentle prodding.
(3) “Flow-through” means a continuous or an intermittent passage of test solution or dilution water through a test chamber or a holding or acclimation tank, with no recycling.
(4) “G1 (Generation 1)” means those mysids which are used to begin the test, also referred to as adults; G2 (Generation 2) are the young produced by G1.
(5) “LC
(6) “Loading” means the ratio of test organism biomass (gram, wet weight) to the volume (liters) of test solution in a test chamber.
(7) “MATC” (Maximum Acceptable Toxicant Concentration) means the maximum concentration at which a chemical can be present and not be toxic to the test organism.
(8) “Retention chamber” means a structure within a flow-through test chamber which confines the test organisms, facilitating observation of test organisms and eliminating washout from test chambers.
(c) Test procedures – (1) Summary of the test. (i) In preparation for the test, the flow of test solution through each chamber is adjusted to the rate desired. The test substance is introduced into each test chamber. The rate at which the test substance is added is adjusted to establish and maintain the desired concentration of test substance in each test chamber. The test is started by randomly introducing mysids acclimated in accordance with the test design into retention chambers within the test and the control chambers. Mysids in the test and control chambers are observed periodically during the test, the dead mysids removed and the findings reported.
(ii) Dissolved oxygen concentration, pH, temperature, salinity, the concentration of test substance and other water quality characteristics are measured at specified intervals in selected test chambers.
(iii) Data collected during the test are used to develop a MATC (Maximum Acceptable Toxicant Concentration) and quantify effects on specific chronic parameters.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted to establish test solution concentrations for the definitive test.
(ii) The mysids should be exposed to a series of widely spaced concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually under static conditions.
(iii) A minimum of 10 mysids should be exposed to each concentration of test substance for a period of time which allows estimation of appropriate chronic test concentrations. No replicates are required and nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to determine concentration-response curves, LC
(ii) A minimum of 40 mysids per concentration shall be exposed to four or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/1). An equal number of mysids shall be placed in two or more replicates. If solvents, solubilizing agents or emulsifiers have to be used, they shall be commonly used carriers and shall not possess a synergistic or antagonistic effect on the toxicity of the test substance. The concentration of solvent should not exceed 0.1 ml/1. The concentration ranges should be selected to determine the concentration response curves, LC
(iii) Every test should include controls consisting of the same dilution water, conditions, procedures and mysids from the same population or culture container, except that none of the chemical is added.
(iv) The dissolved oxygen concentration, temperature, salinity, and pH shall be measured weekly in each chamber.
(v) The test duration is 28 days. The test is unacceptable if more than 20 percent of the control organisms die, appear stressed or are diseased during the test. The number of dead mysids in each chamber shall be recorded on days 7, 14, 21, and 28 of the test. At the time when sexual characteristics are discernible in the mysids (approximately 10 to 12 days in controls; possible delays may occur in mysids exposed to test substances), the number of males and females (identified by ventral brood pouch) in each chamber shall be recorded. Body length (as measured by total midline body length, from the anterior tip of the carapace to the posterior margin of the uropod) shall be recorded for males and females at the time when sex can be determined simultaneously for all mysids in control and treatment groups. This time cannot be specified because of possible delays in sexual maturation of mysids exposed to test substances. A second observation of male and female body lengths shall be conducted on day 28 of the test. To reduce stress on the mysids, body lengths can be recorded by photography through a stereomicroscope with appropriate scaling information. As offspring are produced by the G1 mysids (approximately 13 to 16 days in controls), the young shall be counted and separated into retention chambers at the same test substance concentration as the chambers where they originated. If available prior to termination of the test, observations on the mortality, number of males and females and male and female body length shall be recorded for the G2 mysids. Concentration-response curves, LC
(vi) In addition to death, any abnormal behavior or appearance shall also be reported.
(vii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner or in a way in which appropriate statistical analyses can be used to determined the variation due to placement.
(viii) The concentration of the test substance in the chambers should be measured as often as is feasible during the test. The concentration of test substance shall be measured:
(A) At each test concentration at the beginning of the test and on days 7, 14, 21, and 28; and
(B) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system.
(5) [Reserved]
(6) Analytical measurements – (i) Test chemical. Deionized water should be used in making stock solutions of the test substance. Standard analytical methods should be employed whenever available in performing the analyses. The analytical method used to measure the amount of test substance in a sample shall be validated before beginning the test by appropriate laboratory practices. An analytical method is not acceptable if likely degradation products of the test substance, such as hydrolysis and oxidation products, give positive or negative interferences which cannot be systematically identified and corrected mathematically.
(ii) Numerical. (A) The number of dead mysids, cumulative young per female, and body lengths of male and female mysids shall be recorded during each definitive test. Appropriate statistical analyses shall provide a goodness-of-fit determination for the day 7, 14, 21 and 28 adult (Gl) death concentration-response curves.
(B) A 7-, 14-, 21- and 28-day LC
(d) Test conditions – (1) Test species – (i) Selection. (A) The mysid shrimp, Mysidopsis bahia, is the organism specified for these tests. Juvenile mysids, ≤24 hours old, are to be used to start the test.
(B) Mysids to be used in chronic toxicity tests should originate from laboratory cultures in order to ensure the individuals are of similar age and experimental history. Mysids used for establishing laboratory cultures may be purchased commercially or collected from appropriate natural areas. Because of similarities with other mysid species, taxonomic verification should be obtained from the commercial supplier, by experienced laboratory personnel, or by an outside expert.
(C) Mysids used in a particular test shall be of similar age and be of normal size and appearance for their age.
(D) Mysids shall not be used for a test if they exhibit abnormal behavior, or if they have been used in a previous test, either in a treatment or in a control group.
(ii) Acclimation. (A) Any change in the temperature and chemistry of the water used for holding or culturing the test organisms to those of the test should be gradual. Within a 24-hour period, changes in water temperature should not exceed 1 °C, while salinity changes should not exceed 5 percent.
(B) During acclimation mysids should be maintained in facilities with background colors and light intensities similar to those of the testing areas.
(iii) Care and handling. Methods for the care and handling of mysids such as those described in paragraph (f)(1) of this section can be used during holding, culturing and testing periods.
(iv) Feeding. Mysids should be fed during testing. Any food utilized should support survival, growth and reproduction of the mysids. A recommended food is live Artemia spp. nauplii (approximately 48 hours old).
(2) Facilities – (i) Apparatus. (A) Facilities which may be needed to perform this test include: (1) flow-through or recirculating tanks for holding and acclimating mysids; (2) a mechanism for controlling and maintaining the water temperature during the holding, acclimation and test periods; (3) apparatus for straining particulate matter, removing gas bubbles, or aerating the water, as necessary; and (4) an apparatus for providing a 14-hour light and 10-hour dark photoperiod with a 15- to 30-minute transition period. In addition, flow-through chambers and a test substance delivery system are required. It is recommended that mysids be held in retention chambers within test chambers to facilitate observations and eliminate loss through outflow water.
(B) Facilities should be well ventilated and free of fumes and disturbances that may affect test organisms.
(C) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions.
(ii) Cleaning. Test substance delivery systems and test chambers shall be cleaned before each use following standard laboratory practices.
(iii) Construction materials. (A) Materials and equipment that contact test solutions should be chosen to minimize sorption of test chemicals from the dilution water and should not contain substances that can be leached into aqueous solution in quantities that can affect the test results.
(B) Retention chambers utilized for confinement of test organisms can be constructed with netting material of appropriate mesh size.
(iv) Dilution water. (A) Natural or artificial seawater is acceptable as dilution water if mysids will survive and successfully reproduce in it for the duration of the holding, acclimating and testing periods without showing signs of stress, such as reduced growth and fecundity. Mysids shall be cultured and tested in dilution water from the same origin.
(B) Natural seawater shall be filtered through a filter with a pore size of >20 microns prior to use in a test.
(C) Artificial seawater can be prepared by adding commercially available formulations or by adding specific amounts of reagent-grade chemicals to deionized or glass-distilled water. Deionized water with a conductivity less than 1 µohm/cm at 12 °C is acceptable as the diluent for making artificial seawater. When deionized water is prepared from a ground or surface water source, conductivity and total organic carbon (or chemical oxygen demand) shall be measured on each batch.
(v) Test substance delivery system. Proportional diluters, metering pumps, or other suitable systems should be used to deliver test substance to the test chambers. The system used shall be calibrated before each test. Calibration includes determining the flow rate and the concentration of the test substance in each chamber. The general operation of the test substance delivery system should be checked twice daily during a test. The 24-hour flow rate through a chamber shall be equal to at least 5 times the volume of the chamber. The flow rates should not vary more than 10 percent among chambers or across time.
(3) Test parameters. Environmental parameters of the water contained in test chambers shall be maintained as specified below:
(i) The test temperature shall be 25 °C. Excursions from the test temperature shall be no greater than ±2 °C.
(ii) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test substance. All treatment and control chambers shall be given the same aeration treatment.
(iii) The number of mysids placed in a test solution shall not be so great as to affect results of the test. Loading requirements for the test will vary depending on the flow rate of dilution water. The loading shall not cause the dissolved oxygen concentration to fall below the recommended levels.
(iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15-30 minute transition period.
(v) Salinity of 20 parts per thousand ±3 percent.
(e) Reporting. The sponsor shall submit to the EPA all data developed by the test that are suggestive or predictive of chronic toxicity and all concomitant toxicologic manifestations. In addition to the general reporting requirements prescribed in part 792 – Good Laboratory Practice Standards of this chapter, the reporting of test data shall include the following:
(1) The source of the dilution water, its chemical characteristics (e.g., salinity, pH, etc.) and a description of any pretreatment.
(2) Detailed information about the test organisms, including the scientific name and method of verification, average length, age, source, history, observed diseases, treatments, acclimation procedures and food used.
(3) A description of the test chambers, the depth and volume of solution in the chamber, the way the test was begun (e.g., conditioning, test substance additions, etc.), the number of organisms per treatment, the number of replicates, the loading, the lighting, the test substance delivery system, and the flow rate expressed as volume additions per 24 hours.
(4) The measured concentration of test substance in test chambers at the times designated.
(5) The first time (day) that sexual characteristics can be observed in controls and in each test substance concentration.
(6) The length of time for the appearance of the first brood for each concentration.
(7) The means (average of replicates) and respective 95 percent confidence intervals for:
(i) Body length of males and females at the first observation day (depending on time of sexual maturation) and on day 28.
(ii) Cumulative number of young produced per female on day 28.
(iii) Cumulative number of dead adults on day 7, 14, 21 and 28.
(iv) If available prior to test termination (day 28), effects on G2 mysids (number of males and females, body length of males and females and cumulative mortality).
(8) The MATC is calculated as the geometric mean between the lowest measured test substance concentration that had a significant (P
(9) Concentration-response curves shall be fitted to the cumulative number of adult dead for days 7, 14, 21, and 28. A statistical test of goodness-of-fit shall be performed and the results reported.
(10) An LC
(11) Methods and data records of all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks.
(12) The data records of the holding, acclimation and test temperature and salinity.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency, “Bioassay Procedures for the Ocean Disposal Permit Program,” EPA Report No. 600/9-78-010 (Gulf Breeze, Florida, 1978).
(2) [Reserved]
PART 798 – HEALTH EFFECTS TESTING GUIDELINES
Subparts A-B [Reserved]
Subpart C – Subchronic Exposure
§ 798.2250 Dermal toxicity.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic dermal toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic dermal study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the dermal route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.
(b) Definitions. (1) Subchronic dermal toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by dermal application for part (approximately 10 percent) of a life span.
(2) Dose in a dermal test is the amount of test substance applied to the skin (applied daily in subchronic tests). Dose is expressed as weight of the substance (g, mg) per unit weight of test animal (e.g., mg/kg).
(3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of the weight of a test substance given daily per unit weight of test animal (mg/kg).
(4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of the administered test substance or its metabolites in susceptible tissues.
(c) Principle of the test method. The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose level per unit group, for a period of 90 days. During the period of application the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied and appropriate histopathological examinations carried out.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary.
(e) Test procedures – (1) Animal selection – (i) Species and strain. A mammalian species shall be used for testing. The rat, rabbit, or guinea pig may be used, although the albino rabbit is preferred. The albino rabbit is preferred because of its size, skin permeability, and extensive data base. Commonly used laboratory strains shall be employed. If another mammalian species is used, the tester shall provide justification/reasoning for its selection.
(ii) Age. Young adult animals shall be used. The following weight ranges at the start of the test are suggested in order to provide animals of a size which facilitates the conduct of the test: rats, 200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs, 350 to 450 g.
(iii) Sex. (A) Equal numbers of animals of each sex with healthy skin shall be used at each dose level.
(B) The females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 animals (10 females and 10 males) shall be used at each dose level.
(B) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed before completion of the study.
(2) Control groups. A concurrent control group is required. This group shall be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(3) Satellite group. A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence, of toxic effects for a posttreatment period of appropriate length, normally not less than 28 days.
(4) Dose level and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose-response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data shall be sufficient to produce a dose-response curve.
(ii) The highest dose level should result in toxic effects but not produce severe skin irritation or an incidence of fatalities which would prevent a meaningful evaluation.
(iii) The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure, the lowest dose level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects.
(v) In the low and intermediate groups and in the controls the incidence of fatalities should be low, to permit a meaningful evaluation of the results.
(5) Exposure conditions. The animals are treated with test substance, ideally for at least 6 hours per day on a 7-day per week basis, for a period of 90 days. However, based primarily on practical considerations, application on a 5-day per week basis is considered to be acceptable.
(6) Observation period. (i) Duration of observation shall be at least 90 days.
(ii) Animals in the satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(7) Preparation of animal skin. (i) Shortly before testing, fur shall be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin, which could alter its permeability.
(ii) Not less than 10 percent of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of any covering used.
(iii) When testing solids, which may be pulverized if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of and penetration of the skin by the test substance should be taken into account.
(8) Application of the test substance. (i) The test substance shall be applied uniformly over an area which is approximately 10 percent of the total body surface area. With highly toxic substances, the surface area covered may be less, but as much of the area shall be covered with as thin and uniform a film as possible.
(ii) During the exposure period, the test substance shall be held in contact with the skin with a porous gauze dressing and nonirritating tape. The test site shall be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not a recommended method.
(9) Observation of animals. (i) Each animal shall be observed daily, and if necessary handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree, and duration.
(iv) Cage-side observations shall include, but not be limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.
(v) Animals shall be weighed weekly. Feed consumption shall also be determined weekly if abnormal body weight changes are observed.
(vi) At the end of the study period, all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.
(10) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group:
(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumen blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include: Analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.
(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected all animals should be examined.
(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to exposure to the test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected or observed toxicity.
(11) Gross necropsy. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.
(ii) The liver, kidneys, adrenals, brain, and gonads shall be weighed wet, as soon as possible after dissection, to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs – which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain – including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur – including articular surface); (spinal cord at three levels – cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
(12) Histopathology. The following histopathology shall be performed:
(i) Full histopathology on normal and treated skin and on organs and tissues, listed above, of all animals in the control and high dose groups.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues listed in parenthesis in paragraph (e)(11)(iii) of this section, if indicated by signs of toxicity or expected target organ involvement.
(v) Lungs of animals (rodents) in the low and intermediate dose groups shall be subjected to histopathological examination for evidence of infection, since this provides a convenient assessment of the state of health of the animals.
(vi) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(f) Data and reporting – (1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions, and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of results. The findings of a subchronic dermal toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the observed toxic effects and the necropsy and histopathological findings. The evaluation should include the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effect on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified in the EPA Good Laboratory Practice Standards under 40 CFR part 792, subpart J, the following specific information shall be reported.
(i) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii) Individual animal data. (A) Date of death during the study or whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Draize, J.H. “Dermal toxicity,” Appraisal of Chemicals in Food, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd printing 1975). pp. 46-59.
(2) Fitzhugh, O.G. “Subacute toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd printing 1975). pp. 26-35.
(3) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” a report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(4) World Health Organization. “Part I. Environmental Health Criteria 6,”Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
§ 798.2450 Inhalation toxicity.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a gas, volatile substance, or aerosol/particulate, determination of subchronic inhalation toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic inhalation study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposures by the inhalation route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure. Hazards of inhaled substances are influenced by the inherent toxicity and by physical factors such as volatility and particle size.
(b) Definitions. (1) Subchronic inhalation toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by inhalation for part (approximately 10 percent) of a life span.
(2) Aerodynamic diameter applies to the size of particles of aerosols. It is the diameter of a sphere of unit density which behaves aerodynamically as the particle of the test substance. It is used to compare particles of different size and densities and to predict where in the respiratory tract such particles may be deposited. This term is used in contrast to measured or geometric diameter which is representative of actual diameters which in themselves cannot be related to deposition within the respiratory tract.
(3) The geometric mean diameter or the median diameter is the calculated aerodynamic diameter which divides the particles of an aerosol in half based on the weight of the particles. Fifty percent of the particles by weight will be larger than the median diameter and 50 percent of the particles will be smaller than the median diameter. The median diameter describes the particle size distribution of any aerosol based on the weight and size of the particles.
(4) Inhalable diameter refers to that aerodynamic diameter of a particle which is considered to be inhalable for the organism. It is used to refer to particles which are capable of being inhaled and may be deposited anywhere within the respiratory tract from the trachea to the alveoli. For man, inhalable diameter is considered as 15 micrometers or less.
(5) Dose refers to an exposure level. Exposure is expressed as weight or volume of test substance per volume of air (mg/l), or as parts per million (ppm).
(6) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight or volume of test substance given daily per unit volume of air (mg/l or ppm).
(7) Cumulative toxicity is the adverse effects of repeated doses occuring as a result of prolonged action on, or increased concentration of the administered test substance or its metabolites in susceptible tissues.
(c) Principle of the test method. Several groups of experimental animals are exposed daily for a defined period to the test substance in graduated concentrations, one concentration being used per group, for a period of 90 days. During the period of administration, the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied and at the conclusion of the test, surviving animals are sacrificed and necropsied and appropriate histopathological examinations carried out.
(d) Test procedures – (1) Animal selection – (i) Species and strain. A mammalian species shall be used for testing. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains shall be employed. If another mammalian species is used, the tester shall provide justification/ reasoning for its selection.
(ii) Age. Young adult animals shall be used. At the commencement of the study the weight variation of animals shall not exceed ±20 percent of the mean weight for each sex.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.
(B) Females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 rodents (10 females and 10 males) shall be used for each test group. If another mammalian species is selected (e.g. dog, rabbit, or non-human primate), at least 8 animals (4 males and 4 females) shall be used.
(B) If interim sacrifices are planned, the number of animals shall be increased by the number of animals scheduled to be sacrificed before the completion of the study.
(2) Control groups. A concurrent control group is required. This group shall be an untreated or sham-treated control group. Except for treatment with the test substance, animals in the control group shall be handled in a manner identical to the test group animals. Where a vehicle is used to help generate an appropriate concentration of the substance in the atmosphere, a vehicle control group shall be used. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(3) Satellite group. A satellite group of 20 animals (10 animals per sex) may be treated with the high concentration level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.
(4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a concentration-response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 concentration levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Concentrations should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a concentration-response curve.
(ii) The highest concentration should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation.
(iii) The lowest concentration should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest concentration should exceed this.
(iv) Ideally, the intermediate concentration level(s) should produce minimal observable toxic effects. If more than one intermediate concentration level is used, the concentrations should be spaced to produce a gradation of toxic effects.
(v) In the low and intermediate groups and in the controls the incidence of fatalities should be low, to permit a meaningful evaluation of the results.
(vi) In the case of potentially explosive test substances, care should be taken to avoid generating explosive concentrations.
(5) Exposure conditions. The animals should be exposed to the test substance, ideally for 6 hours per day on a 7-day per week basis, for a period of 90 days. However, based primarily on practical considerations, exposure on a 5-day-per-week basis for 6 hours per day is the minimum acceptable exposure period.
(6) Observation period. (i) Duration of observation shall be for at least 90 days.
(ii) Animals in a satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(7) Inhalation exposure. (i) The animals shall be tested in inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour and ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber. Oronasal or head-only exposure may be used if it is desirable to avoid concurrent exposure by the dermal or oral routes.
(ii) A dynamic inhalation system with a suitable flow control system shall be used. The rate of air flow shall be adjusted to ensure that conditions throughout the exposure chamber are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas.
(iii) The temperature at which the test is performed should be maintained at 22 °C (±2°). Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(8) Physical measurements. Measurements or monitoring shall be made of the following:
(i) The rate of air flow shall be monitored continuously and recorded at least every 30 minutes.
(ii) The actual concentrations of the test substance shall be measured in the breathing zone. During the exposure period the actual concentrations of the test substance shall be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis, and recorded at least at the beginning, at an intermediate time, and at the end of the exposure period.
(iii) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. During exposure, analysis shall be conducted as often as necessary to determine the consistency of particle size distribution.
(iv) Temperature and humidity shall be monitored continuously but shall be recorded at least every 30 minutes.
(9) Feed and water during exposure period. Feed shall be withheld during exposure. Water may also be withheld during exposure.
(10) Observation of animals. (i) Each animal shall be observed daily and, if necessary, handled to appraise its physical condition.
(ii) Additional observations should be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed including the time of onset, the degree, and duration.
(iv) Cage-side observations should include, but not be limited to, changes in the skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.
(v) Animals shall be weighed weekly. Feed consumption shall also be determined weekly if abnormal body weight changes are observed.
(vi) At the end of the study period all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.
(11) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group:
(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations: calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase, (now known as serum alanine aminotransferase), serum glutamic-oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include: Analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.
(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected, all animals shall be examined:
(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to exposure to the test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected and/or observed toxicity.
(12) Gross pathology. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices and the cranial, thoracic, and abdominal cavities and their contents.
(ii) At least the liver, kidneys, adrenals, brain, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs – which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain – including sections of medulla/pons cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur – including articular surface); (spinal cord at three levels – cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
(13) Histopathology. The following histopathology shall be performed:
(i) Full histopathology on the respiratory tract and other organs and tissues, listed above, of all animals in the control and high dose groups.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues mentioned in brackets (listed above) if indicated by signs of toxicity or target organ involvement.
(v) Lungs of animals (rodents) in the low and intermediate dose groups shall also be subjected to histopathological examination, primarily for evidence of infection since this provides a convenient assessment of the state of health of the animals.
(vi) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(e) Data and reporting – (1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions, and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of results. The findings of the subchronic inhalation toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the observed toxic effects and the necropsy and histopathological findings. The evaluation will include the relationship between the concentration of the test substance and duration of exposure, and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus, including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air, and the method of housing animals in a test chamber.
(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean values and measure of variability (e.g., standard deviation) and shall include:
(A) Airflow rates through the inhalation equipment.
(B) Temperature and humidity of air.
(C) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
(D) Actual concentration in test breathing zone.
(E) Particle size distribution (e.g., median aerodynamic diameter of particles with standard deviation from the mean).
(iii) Group animal data. Tabulation of toxic response data by species, strain, sex, and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(iv) Individual animal data. (A) Date of death during the study or whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Cage, J.C. “Experimental Inhalation Toxicology,” Methods in Toxicology. Ed. G.E. Paget. (Philadelphia: F.A. Davis Co. 1970, pp. 258-277.
(2) Casarett, L.J., Doull, J. “Chapter 9.” Toxicology: The Basic Science of Poisons (New York: Macmillan Publishing Co. Inc. 1975).
(3) MacFarland, H.N. “Respiratory Toxicology,” Essays in Toxicology. Ed. W.J. Hayes. Vol. 7 (New York: Academic Press, 1976) pp. 121-154.
(4) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” a report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(5) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
§ 798.2650 Oral toxicity.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic oral toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic oral study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.
(b) Definitions. (1) Subchronic oral toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the oral route for a part (approximately 10 percent) of a life span.
(2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of test animal (e.g., mg/kg), or as weight of test substance per unit weight of food or drinking water.
(3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of the weight of a substance given daily per unit weight of test animal (mg/kg). When administered to animals in food or drinking water the no-observed-effect level is expressed as mg/kg of food or mg/ml of water.
(4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissue.
(c) Principle of the test method. The test substance is administered orally in graduated daily doses to several groups of experimental animals, one dose level per group, for a period of 90 days. During the period of administration the animals are observed daily to detect signs of toxicity. Animals which die during the period of administration are necropsied. At the conclusion of the test all animals are necropsied and histo-pathological examinations carried out.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary.
(e) Test procedures – (1) Animal selection – (i) Species and strain. A mammalian species shall be used for testing. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains shall be employed. The commonly used nonrodent species is the dog, preferably of a defined breed; the beagle is frequently used. If other mammalian species are used, the tester shall provide justification/reasoning for his or her selection.
(ii) Age – (A) General. Young adult animals shall be employed. At the commencement of the study the weight variation of animals used shall not exceed ±20 percent of the mean weight for each sex.
(B) Rodents. Dosing shall begin as soon as possible after weaning, ideally before the rats are 6, and in any case, not more than 8 weeks old.
(C) Non-rodent. In the case of the dog, dosing shall commence after acclimatization, preferably at 4 to 6 months and not later than 9 months of age.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.
(B) The females shall be nulliparous and nonpregnant.
(iv) Numbers – (A) Rodents. At least 20 animals (10 females and 10 males) shall be used at each dose level.
(B) Non-rodents. At least eight animals (four females and four males) shall be used at each dose level.
(C) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed before the completion of the study.
(2) Control groups. A concurrent control group is required. This group shall be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(3) Satellite group. (Rodent) A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.
(4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.
(ii) The highest dose level in rodents should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation; for non-rodents there should be no fatalities.
(iii) The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest dose level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate dose groups and in the controls should be low, to permit a meaningful evaluation of the results; for non-rodents, there should be no fatalities.
(5) Exposure conditions. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of 90 days. However, based primarily on practical considerations, dosing in gavage or capsule studies on a 5-day per week basis is considered to be acceptable.
(6) Observation period. (i) Duration of observation shall be for at least 90 days.
(ii) Animals in the satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(7) Administration of the test substance. (i) The test substance may be administered in the diet or in capsules. In addition, for rodents it may also be administered by gavage or in the drinking water.
(ii) All animals shall be dosed by the same method during the entire experimental period.
(iii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, ideally it should not elicit important toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the usage of an aqueous solution be considered first, followed by consideration of a solution of oil and then by possible solution in other vehicles.
(iv) For substances of low toxicity, it is important to ensure that when administered in the diet the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals’ body weight shall be used; the alternative used shall be specified.
(v) For a substance administered by gavage or capsule, the dose shall be given at approximately the same time each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body weight.
(8) Observation of animals. (i) Each animal shall be observed daily and, if necessary, handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed including the time of onset, degree and duration.
(iv) Cage-side observations shall include, but not be limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.
(v) Measurements shall be made weekly of feed consumption or water consumption when the test substance is administered in the feed or drinking water, respectively.
(vi) Animals shall be weighed weekly.
(vii) At the end of the 90-day period all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.
(9) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group for rodents and all animals when non-rodents are used as test animals.
(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include: Analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.
(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected, all animals should be examined.
(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to the administration of the test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected and or observed toxicity.
(10) Gross necropsy. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
(ii) At least the liver, kidneys, adrenals, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs – which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain – including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur – including articular surface); (spinal cord at three levels – cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands); and (rodent-zymbal glands).
(11) Histopathology. The following histopathology shall be performed:
(i) Full histopathology on the organs and tissues, listed above, of all rodents in the control and high dose groups, all non-rodents, and all rodents that died or were killed during the study.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues mentioned in brackets (listed above) if indicated by signs of toxicity of target organ involvement.
(v) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents shall be made for evidence of infection since this provides a convenient assessment of the state of health of the animals.
(vi) When a satellite group is used (rodents), histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(f) Data and reporting – (1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of the study results. (i) The findings of a subchronic oral toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects and the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(3) Test report. In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii) Individual animal data. (A) Date of death during the study or whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Boyd, E.M. “Chapter 14 – Pilot Studies, 15 – Uniposal Clinical Parameters, 16 – Uniposal Autopsy Parameters.” Predictive Toxicometrics. (Baltimore: Williams and Wilkins, 1972).
(2) Fitzhugh, O.G. “Subacute Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975) pp. 26-35.
(3) Food Safety Council. “Subchronic Toxicity Studies,” Proposed System for Food Safety Assessment. (Columbia: Food Safety Council, 1978) pp. 83-96.
(4) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” a report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(5) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
Subpart D – Chronic Exposure
§ 798.3260 Chronic toxicity.
(a) Purpose. The objective of a chronic toxicity study is to determine the effects of a substance in a mammalian species following prolonged and repeated exposure. Under the conditions of the chronic toxicity test, effects which require a long latency period or which are cumulative should become manifest. The application of this guideline should generate data on which to identify the majority of chronic effects and shall serve to define long term dose-response relationships. The design and conduct of chronic toxicity tests should allow for the detection of general toxic effects, including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathology) effects.
(b) Test procedures – (1) Animal selection – (i) Species and strain. Testing should be performed with two mammalian species, one a rodent and another a non-rodent. The rat is the preferred rodent species and the dog is the preferred non-rodent species. Commonly used laboratory strains should be employed. If other mammalian species are used, the tester should provide justification/reasoning for their selection.
(ii) Age. (A) Dosing of rats should begin as soon as possible after weaning, ideally before the rats are 6, but in no case more than 8 weeks old.
(B) Dosing of dogs should begin between 4 and 6 months of age and in no case later than 9 months of age.
(C) At commencement of the study the weight variation of animals used should not exceed ±20 percent of the mean weight for each sex.
(iii) Sex. (A) Equal numbers of animals of each sex should be used at each dose level.
(B) The females should be nulliparous and non-pregnant.
(iv) Numbers. (A) For rodents, at least 40 animals (20 females and 20 males) and for non-rodents (dogs) at least 8 animals (4 females and 4 males) should be used at each dose level.
(B) If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed during the course of the study.
(C) The number of animals at the termination of the study must be adequate for a meaningful and valid statistical evaluation of chronic effects.
(2) Control groups. (i) A concurrent control group is suggested. This group should be an untreated or sham treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are strongly suggested.
(ii) In special circumstances such as in inhalation studies involving aerosols or the use of an emulsifier of uncharacterized biological activity in oral studies, a concurrent negative control group should be utilized. The negative control group should be treated in the same manner as all other test animals except that this control group should not be exposed to either the test substance or any vehicle.
(3) Dose levels and dose selections. (i) In chronic toxicity tests, it is necessary to have a dose-response relationship as well as a no-observed-toxic-effect level. Therefore, at least three dose levels should be used in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of effects.
(ii) The high dose level in rodents should elicit some signs of toxicity without causing excessive lethality; for non-rodents, there should be signs of toxicity but there should be no fatalities.
(iii) The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest dose level should exceed this even though this dose level may result in some signs of toxicity.
(iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose level should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate dose groups and in the controls should be low to permit a meaningful evaluation of the results. For non-rodents, there should be no fatalities.
(4) Exposure conditions. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of at least 12 months. However, based primarily on practical considerations, dosing on a 5-day per week basis is considered to be acceptable.
(5) Observation period. Duration of observation should be for at least 12 months, and may be concurrent with or subsequent to dosing. If there is a post-exposure observation period, an interim sacrifice should be performed on no fewer than half of the animals of each sex at each dose level immediately upon termination of exposure.
(6) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals should receive the test substance in their diet, dissolved in drinking water, or given by gavage or capsule for a period of at least 12 months.
(B) If the test substance is administered in the drinking water, or mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should not exceed 5 percent.
(ii) Dermal studies. (A) The animals are treated by topical application with the test substance, ideally for at least 6 hours per day.
(B) Fur should be clipped from the dorsal area of the trunk of the test animals. Care must be taken to avoid abrading the skin which could alter its permeability.
(C) The test substance should be applied uniformly over a shaved area which is approximately 10 percent of the total body surface area. With highly toxic substances, the surface area covered may be less, but as much of the area should be covered with as thin and uniform a film as possible.
(D) During the exposure period, the test substance may be held if necessary, in contact with the skin with a porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance.
(iii) Inhalation studies. (A) The animals should be tested with inhalation equipment designed to sustain a dynamic air flow of 12 to 15 air changes per hour, ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. As a general rule to ensure stability of a chamber atmosphere, the total “volume” of the test animals should not exceed 5 percent of the volume of the test chamber. Alternatively, oro-nasal, head-only or whole body individual chamber exposure may be used.
(B) The temperature at which the test is performed should be maintained at 22 °C (±2°). Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(C) Feed and water should be withheld during each daily 6 hour exposure period.
(D) A dynamic inhalation system with a suitable analytical concentration control system should be used. The rate of air flow should be adjusted to ensure that conditions throughout the equipment are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas.
(7) Observation of animals. (i) Each animal should be handled and its physical condition appraised at least once each day.
(ii) Additional observations should be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrific of weak or moribund animals).
(iii) Clinical signs of toxicity including suspected tumors and mortality should be recorded as they are observed, including the time of onset, the degree and duration.
(iv) Cage-side observations should include, but not be limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.
(v) Body weights should be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighings to facilitate monitoring of health status.
(vi) When the test substance is administered in the feed or drinking water, measurements of feed or water consumption, respectively, should be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise.
(vii) At the end of the study period all survivors should be sacrificed. Moribund animals should be removed and sacrificed when noticed.
(8) Physical measurements. For inhalation studies, measurements or monitoring should be made of the following:
(i) The rate of air flow should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the test substance should be held as constant as practicable, monitored continuously and measured at least three times during the test period: at the beginning, at an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size analysis should be performed to establish the stability of aerosol concentrations. During exposure, analysis should be conducted as often as necessary to determine the consistency of particle size distribution and homogeneity of the exposure stream.
(iv) Temperature and humidity should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. The following examinations should be made on at least 10 rats of each sex per dose and on all non-rodents.
(i) Certain hematology determinations (e.g., hemoglobin content, packed cell volume, total red blood cells, total white blood cells, platelets, or other measures of clotting potential) should be performed at termination and should be performed at 3 months, 6 months and at approximately 6 month intervals thereafter (for studies extending beyond 12 months) on blood samples collected from all non-rodents and from 10 rats per sex of all groups. These collections should be from the same animals at each interval. If clinical observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals should be performed. A differential blood count should be performed on samples from those animals in the highest dosage group and the controls. Differential blood counts should be performed for the next lower group(s) if there is a major discrepancy between the highest group and the controls. If hematological effects were noted in the subchronic test, hematological testing should be performed at 3, 6, 12, 18, and 24 months for a two year study and at 3, 6, and 12 months for a 1-year study.
(ii) Certain clinical biochemistry determinations on blood should be carried out at least three times during the test period: just prior to initiation of dosing (base line data), near the middle and at the end of the test period. Blood samples should be drawn for clinical chemistry measurements from all non-rodents and at least ten rodents per sex of all groups; if possible, from the same rodents at each time interval. Test areas which are considered appropriate to all studies: electrolyte balance, carbohydrate metabolism and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity. Suggested chemical determinations: calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, blood urea nitrogen, albumen, blood creatinine, creatinine phosphokinase, total cholesterol, total bilirubin and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methemoglobin and cholinesterase activity. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects.
(iii) Urine samples from rodents at the same intervals as the hematological examinations under paragraph (b)(9)(i) of this section should be collected for analysis. The following determinations should be made from either individual animals or on a pooled sample/sex/group for rodents: appearance (volume and specific gravity), protein, glucose, ketones, bilirubin, occult blood (semi-quantitatively); and microscopy of sediment (semi-quantitatively).
(iv) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study. If changes in eyes are detected all animals should be examined.
(10) Gross necropsy. (i) A complete gross examination should be performed on all animals, including those which died during the experiment or were killed in moribund conditions.
(ii) The liver, kidneys, adrenals, brain and gonads should be weighed wet, as soon as possible after dissection to avoid drying. For these organs, at least 10 rodents per sex per group and all non-rodents should be weighed.
(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination: All gross lesions and tumors; brain – including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; sternum and/or femur with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles; female mammary gland; aorta; gall bladder (if present); skin; musculature; peripheral nerve; spinal cord at three levels – cervical, midthoracic, and lumbar; and eyes. In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from sites of skin painting should be examined and preserved.
(iv) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is considered essential for appropriate and valid histopathological examination.
(v) If other clinical examinations are carried out, the information obtained from these procedures should be available before microscopic examination, since they may provide significant guidance to the pathologist.
(11) Histopathology. (i) The following histopathology should be performed:
(A) Full histopathology on the organs and tissues, listed above, of all non-rodents, of all rodents in the control and high dose groups and of all rodents that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents should be made for evidence of infection since this provides an assessment of the state of health of the animals.
(ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level should be examined for complete histopathology.
(iii) In case the results of an experiment give evidence of substantial alteration of the animals’ normal longevity or the induction of effects that might affect a toxic response, the next lower dose level should be examined fully, as described under paragraph (b)(11)(i) of this section.
(iv) An attempt should be made to correlate gross observations with microscopic findings.
(c) Data and reporting – (1) Treatment of results. (i) Data should be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of study results. (i) The findings of a chronic toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence, incidence and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(3) Test report. (i) In addition to the reporting requirements as specified under 40 CFR part 792 subpart J, the following specific information should be reported:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(1) Number of animals dying.
(2) Number of animals showing signs of toxicity.
(3) Number of animals exposed.
(B) Individual animal data. (1) Time of death during the study or whether animals survived to termination.
(2) Time of observation of each abnormal sign and its subsequent course.
(3) Body weight data.
(4) Feed and water consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
(6) Hematological tests employed and all results.
(7) Clinical biochemistry tests employed and all results.
(8) Necropsy findings.
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of results, where appropriate.
(ii) In addition, for inhalation studies the following should be reported:
(A) Test conditions. (1) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
(B) Exposure data. These should be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
(4) Actual concentration in test breathing zone.
(5) Particle size distribution (e.g., median aerodynamic diameter of particles with standard deviation from the mean).
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) Benitz, K.F. “Measurement of Chronic Toxicity,” Methods of Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications, 1970) pp. 82-131.
(2) D’Aguanno, W. “Drug Safety Evaluation – Pre-Clinical Considerations,” Industrial Pharmacology: Neuroleptics. Vol. I, Ed. S. Fielding and H. Lal. (Mt. Kisco: Futura Publishing Co. 1974) pp. 317-332.
(3) Fitzhugh, O.G. Third Printing: 1975. “Chronic Oral Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975) pp. 36-45.
(4) Goldenthal, E.I., D’Aguanno, W. “Evaluation of Drugs,” Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975) pp. 60-67.
(5) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” a report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(6) National Center for Toxicological Research. “Appendix B,” Report of Chronic Studies Task Force Committee, April 13-21, 1972. (Rockville: National Center for Toxicological Research, 1972).
(7) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal of Environmental Pathology and Toxicology, 1:161-182 (1977).
(8) Schwartz, E. “Toxicology of Neuroleptic Agents,” Industrial Pharmacology: Neuroleptics Ed. S. Fielding and H. Lal. (Mt. Kisco, Futura Publishing Co., 1974) pp. 203-221.
(9) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (1977).
(10) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,” WHO Technical Report Series No. 563. (Geneva: World Health Organization, 1975).
(11) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
(12) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,” WHO Technical Report Series No. 341. (Geneva: World Health Organization, 1966).
§ 798.3300 Oncogenicity.
(a) Purpose. The objective of a long-term oncogenicity study is to observe test animals for a major portion of their life span for the development of neoplastic lesions during or after exposure to various doses of a test substance by an appropriate route of administration.
(b) Test procedures – (1) Animal selection – (i) Species and strain. A compound of unknown activity shall be tested on two mammalian species. Rats and mice are the species of choice because of their relatively short life spans, the limited cost of their maintenance, their widespread use in pharmacological and toxicological studies, their susceptibility to tumor induction, and the availability of inbred or sufficiently characterized strains. Commonly used laboratory strains shall be employed. If other species are used, the tester shall provide justification/reasoning for their selection.
(ii) Age. (A) Dosing of rodents shall begin as soon as possible after weaning, ideally before the animals are 6 weeks old, but in no case more than 8 weeks old.
(B) At commencement of the study, the weight variation of animals used shall not exceed ±20 percent of the mean weight for each sex.
(C) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii) Sex. (A) Animals of each sex shall be used at each dose level.
(B) The females shall be nulliparous and non-pregnant.
(iv) Numbers. (A) For rodents, at least 100 animals (50 females and 50 males) shall be used at each dose level and concurrent control.
(B) If interim sacrifices are planned the number shall be increased by the number of animals scheduled to be sacrificed during the course of the study.
(C) The number of animals at the termination of the study should be adequate for a meaningful and valid statistical evaluation of long term exposure. For a valid interpretation of negative results, it is essential that survival in all groups does not fall below 50 percent at the time of termination.
(2) Control groups. (i) A concurrent control group is required. This group shall be an untreated or sham treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(ii) In special circumstances such as in inhalation studies involving aerosols or the use of an emulsifier of uncharacterized biological activity in oral studies, a concurrent negative control group shall be utilized. The negative control group shall be treated in the same manner as all other test animals except that this control group shall not be exposed to either the test substance or any vehicle.
(iii) The use of historical control data (i.e., the incidence of tumors and other suspect lesions normally occurring under the same laboratory conditions and in the same strain of animals employed in the test) is desirable for assessing the significance of changes observed in exposed animals.
(3) Dose levels and dose selection. (i) For risk assessment purposes, at least 3 dose levels shall be used, in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of chronic effects.
(ii) The high dose level should elicit signs of minimal toxicity without substantially altering the normal life span.
(iii) The lowest dose should not interfere with normal growth, development and longevity of the animal; and it should not otherwise cause any indication of toxicity. In general, this should not be lower than ten percent of the high dose.
(iv) The intermediate dose(s) should be established in a mid-range between the high and low doses, depending upon the toxicokinetic properties of the chemical, if known.
(v) The selection of these dose levels should be based on existing data, preferably on the results of subchronic studies.
(4) Exposure conditions. The animals are dosed with the test substance ideally on a 7 day per week basis over a period of at least 24 months for rats, and 18 months for mice. However, based primarily on practical considerations, dosing on a 5 day per week basis is considered to be acceptable.
(5) Observations period. It is necessary that the duration of an oncogenicity test comprise the majority of the normal life span of the strain of animals to be used. This time period shall not be less than 24 months for rats and 18 months for mice, and ordinarily not longer than 30 months for rats and 24 months for mice. For longer time periods, and where any other species are used, consultation with the Agency in regard to the duration of the test is advised.
(6) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals shall receive the test substance in their diet, dissolved in drinking water at levels that do not exceed the maximum solubility of the test chemical under testing condition.
(B) If the test substance is administered in the drinking water, or mixed in the diet, exposure shall be continuous.
(C) For a diet mixture, the highest concentration should not exceed 5 percent.
(ii) Dermal studies. (A) The animals are treated by topical application with the test substance, ideally for at least 6 hours per day.
(B) Fur should be clipped from the dorsal area of the trunk of the test animals. Care should be taken to avoid abrading the skin which could alter its permeability.
(C) The test substance shall be applied uniformly over a shaved area which is approximately 10 percent of the total body surface area. With highly toxic substances, the surface area covered may be less, but as much of the area shall be covered with as thin and uniform a film as possible.
(D) During the exposure period, the test substance may be held, if necessary, in contact with the skin with a porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance.
(iii) Inhalation studies. (A) The animals shall be tested with inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour, ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber. Alternatively, oro-nasal, head-only, or whole-body individual chamber exposure may be used.
(B) The temperature at which the test is performed should be maintained at 22 °C (±2°). Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g. tests of aerosols, use of water vehicle) this may not be practicable.
(C) Feed and water shall be withheld during each daily 6-hour exposure period.
(D) A dynamic inhalation system with a suitable flow control system shall be used. The rate of air flow shall be adjusted to ensure that conditions throughout the equipment are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas.
(7) Observations of animals. (i) Each animal shall be observed daily and if necessary should be handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Clinical signs and mortality shall be recorded for all animals. Special attention should be paid to tumor development. The day of onset, location, dimensions, appearance and progression of each grossly visible or palpable tumor shall be recorded.
(iv) Body weights shall be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighings to facilitate monitoring of health status.
(v) When the test substance is administered in the feed or drinking water, measurements of feed or water consumption, respectively, shall be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise.
(vi) At the end of the study period all survivors are sacrificed. Moribund animals shall be removed and sacrificed when noticed.
(8) Physical measurements. For inhalation studies, measurements or monitoring should be made of the following:
(i) The rate of air flow shall be monitored continuously and recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the test substance shall be held as constant as practicable, monitored continuously and recorded at least three times during the test period: at the beginning, at an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. During exposure, analyses shall be conducted as often as necessary to determine the consistency of particle size, distribution, and homogeneity of the exposure stream.
(iv) Temperature and humidity shall be monitored continuously, but shoud be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. At 12 months, 18 months, and at sacrifice, a blood smear shall be obtained from all animals. A differential blood count shall be performed on blood smears from those animals in the highest dosage group and the controls. If these data, or data from the pathological examination indicate a need, then the 12- and 18-month blood smears from other dose levels shall also be examined. Differential blood counts shall be performed for the next lower group(s) if there is a major discrepancy between the highest group and the controls. If clinical observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals shall be performed.
(10) Gross necropsy. (i) A complete gross examination shall be performed on all animals, including those which died during the experiment or were killed in moribund conditions.
(ii) The following organs and tissues or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions and tumors of all animals shall be preserved; brain – including sections of medulla/pons, cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; spinal cord at three levels – cervical, midthoracic and lumbar; sternum and/or femur with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); mammary gland; skin; musculature; peripheral nerve; and eyes. In inhalation studies, the entire respiratory tract shall be preserved, including nasal cavity, pharynx, larynx and paranasal sinuses. In dermal studies, skin from sites of skin painting shall be examined and preserved.
(iii) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is required for appropriate and valid histopathological examination.
(iv) If other clinical examinations are carried out, the information obtained from these procedures shall be available before microscopic examination, since they may provide significant guidance to the pathologist.
(11) Histopathology. (i) The following histopathology shall be performed:
(A) Full histopathology on organs and tissues listed above of all animals in the control and high dose groups and all animals that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If a significant difference is observed in hyperplastic, pre-neoplastic or neoplastic lesions between the highest dose and control groups, microscopic examination shall be made on that particular organ or tissue of all animals in the study.
(iii) If excessive early deaths or other problems occur in the high dose group, compromising the significance of the data, the next lower dose level shall be examined for complete histopathology.
(iv) In case the results of an experiment give evidence of substantial alteration of the animals’ normal longevity or the induction of effects that might affect a neoplastic response, the next lower dose level shall be examined fully as described in this section.
(v) An attempt shall be made to correlate gross observations with microscopic findings.
(c) Data and reporting – (1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, shall be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods shall be selected during the design of the study.
(2) Evaluation of study results. (i) The findings of an oncogenic toxicity study shall be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation shall include the relationship between the dose of the test substance and the presence, incidence and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(iii) In order for a negative test to be acceptable, it shall meet the following criteria: no more than 10 percent of any group is lost due to autolysis, cannibalism, or management problems; and survival in each group should be no less than 50 percent at 18 months for mice and hamsters and at 24 months for rats.
(3) Test report. (i) In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(1) Number of animals dying.
(2) Number of animals showing signs of toxicity.
(3) Number of animals exposed.
(B) Individual animal data. (1) Time of death during the study or whether animals survived to termination.
(2) Time of observation of each abnormal sign and its subsequent course.
(3) Body weight data.
(4) Feed and water consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
(6) Hematological tests employed and all results.
(7) Clinical biochemistry tests employed and all results.
(8) Necropsy findings.
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of results, where appropriate.
(11) Historical control data, if taken into account.
(ii) In addition, for inhalation studies the following shall be reported:
(A) Test conditions. (1) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(B) Exposure data. These shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and shall include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
(4) Actual concentration in test breathing zone.
(5) Particle size distribution (e.g., median aerodynamic diameter of particles with standard deviation from the mean).
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and Welfare. (Canada: Department of Health and Welfare, 1975).
(2) Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation: Panel on Carcinogenesis. “Report on Cancer Testing in the Safety of Food Additives and Pesticides,” Toxicology and Applied Pharmacology. 20:419-438 (1971).
(3) International Union Against Cancer. “Carcinogenicity Testing,” IUCC Technical Report Series. Vol. 2., Ed. I. Berenblum. (Geneva: International Union Against Cancer, 1969).
(4) Leong, B.K.J., Laskin, S. “Number and Species of Experimental Animals for Inhalation Carcinogenicity Studies” Paper presented at Conference on Target Organ Toxicity, September 1975, Cincinnati, Ohio.
(5) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances.” A report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(6) National Cancer Institute. Report of the Subtask Group on Carcinogen Testing to the Interagency Collaborative Group on Environmental Carcinogenesis. (Bethesda: United States National Cancer Institute, 1976).
(7) National Center for Toxicological Research. “Appendix B,” Report of Chronic Studies Task Force Committee. April 13-21 (Rockville: National Center for Toxicological Research, 1972).
(8) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal of Environmental Pathology and Toxicology. 1:161-182 (1977).
(9) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,” Advances in Modern Toxicology Vol. 3, Ed. Kraybill and Mehlman. (Washington, DC: Hemisphere Publishing Corporation, 1977) pp. 87-171.
(10) Sontag, J.M., Page N.P., Saffiotti, U. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1. (Bethesda: United States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program, 1976).
(11) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (1977).
(12) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,” WHO Technical Report Series No. 426. (Geneva: World Health Organization, 1969).
(13) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
§ 798.3320 Combined chronic toxicity/oncogenicity.
(a) Purpose. The objective of a combined chronic toxicity/oncogenicity study is to determine the effects of a substance in a mammalian species following prolonged and repeated exposure. The application of this guideline should generate data which identify the majority of chronic and oncogenic effects and determine dose-response relationships. The design and conduct should allow for the detection of neoplastic effects and a determination of oncogenic potential as well as general toxicity, including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathology) effects.
(b) Test procedures – (1) Animal selection – (i) Species and strain. Preliminary studies providing data on acute, subchronic, and metabolic responses should have been carried out to permit an appropriate choice of animals (species and strain). As discussed in other guidelines, the mouse and rat have been most widely used for assessment of oncogenic potential, while the rat and dog have been most often studied for chronic toxicity. The rat is the species of choice for combined chronic toxicity and oncogenicity studies. The provisions of this guideline are designed primarily for use with the rat as the test species. If other species are used, the tester should provide justification/reasoning for their selection. The strain selected should be susceptible to the oncogenic or toxic effect of the class of substances being tested, if known, and provided it does not have a spontaneous background too high for meaningful assessment. Commonly used laboratory strains should be employed.
(ii) Age. (A) Dosing of rats should begin as soon as possible after weaning, ideally before the rats are 6 weeks old, but in no case more than 8 weeks old.
(B) At commencement of the study, the weight variation of animals used should not exceed ±20 percent of the mean weight for each sex.
(C) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex should be used at each dose level.
(B) The females should be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 females and 50 males) should be used at each dose level and concurrent control for those groups not intended for early sacrifice. At least 40 rodents (20 females and 20 males) should be used for satellite dose group(s) and the satellite control group. The purpose of the satellite group is to allow for the evaluation of pathology other than neoplasia.
(B) If interim sacrifices are planned, the number of animals should be increased by the number of animals scheduled to be sacrificed during the course of the study.
(C) The number of animals at the termination of each phase of the study should be adequate for a meaningful and valid statistical evaluation of long term exposure. For a valid interpretation of negative results, it is essential that survival in all groups not fall below 50 percent at the time of termination.
(2) Control groups. (i) A concurrent control group (50 females and 50 males) and a satellite control group (20 females and 20 males) are recommended. These groups should be untreated or sham treated control groups or, if a vehicle is used in administering the test substance, vehicle control groups. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are recommended. Animals in the satellite control group should be sacrificed at the same time the satellite test group is terminated.
(ii) In special circumstances such as inhalation studies involving aerosols or the use of an emulsifier of uncharacterized biological activity in oral studies, a concurrent negative control group should be utilized. The negative control group should be treated in the same manner as all other test animals, except that this control group should not be exposed to the test substance or any vehicle.
(iii) The use of historical control data (i.e., the incidence of tumors and other suspect lesions normally occuring under the same laboratory conditions and in the same strain of animals employed in the test) is desirable for assessing the significance of changes observed in exposed animals.
(3) Dose levels and dose selection. (i) For risk assessment purposes, at least three dose levels should be used, in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of effects.
(ii) The highest dose level in rodents should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors.
(iii) The lowest dose level should produce no evidence of toxicity. Where there is a usable estimation of human exposure, the lowest dose level should exceed this even though this dose level may result in some signs of toxicity.
(iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used the dose levels should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate dose groups and in the controls should be low to permit a meaningful evaluation of the results.
(vi) For chronic toxicological assessment, a high dose treated satellite and a concurrent control satellite group should be included in the study design. The highest dose for satellite animals should be chosen so as to produce frank toxicity, but not excessive lethality, in order to elucidate a chronic toxicological profile of the test substance. If more than one dose level is selected for satellite dose groups, the doses should be spaced to produce a gradation of toxic effects.
(4) Exposure conditions. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of at least 24 months for rats, and 18 months for mice and hamsters, except for the animals in the satellite groups which should be dosed for 12 months.
(5) Observation period. It is necessary that the duration of the oncogenicity test comprise the majority of the normal life span of the animals to be used. It has been suggested that the duration of the study should be for the entire lifetime of all animals. However, a few animals may greatly exceed the average lifetime and the duration of the study may be unnecessarily extended and complicate the conduct and evaluation of the study. Rather, a finite period covering the majority of the expected life span of the strain is preferred since the probability is high that, for the great majority of chemicals, induced tumors will occur within such an observation period. The following guidelines are recommended:
(i) Generally, the termination of the study should be at 18 months for mice and hamsters and 24 months for rats; however, for certain strains of animals with greater longevity and/or low spontaneous tumor rate, termination should be at 24 months for mice and hamsters and at 30 months for rats. For longer time periods, and where any other species are used, consultation with the Agency in regard to duration of the test is advised.
(ii) However, termination of the study is acceptable when the number of survivors of the lower doses or of the control group reaches 25 percent. In the case where only the high dose group dies prematurely for obvious reasons of toxicity, this should not trigger termination of the study.
(iii) The satellite groups and the concurrent satellite control group should be retained in the study for at least 12 months. These groups should be scheduled for sacrifice for an estimation of test-substance-related pathology uncomplicated by geriatric changes.
(6) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals should receive the test substance in their diet, dissolved in drinking water, or given by gavage or capsule for a period of at least 24 months for rats and 18 months for mice and hamsters.
(B) If the test substance is administered in the drinking water, or mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should not exceed 5 percent.
(ii) Dermal studies. (A) The animals are treated by topical application with the test substance, ideally for at least 6 hours per day.
(B) Fur should be clipped from the dorsal area of the trunk of the test animals. Care should be taken to avoid abrading the skin which could alter its permeability.
(C) The test substance should be applied uniformly over a shaved area which is approximately 10 percent of the total body surface area. With highly toxic substances, the surface area covered may be less, but as much of the area as possible should be covered with as thin and uniform a film as possible.
(D) During the exposure period, the test substance may be held, if necessary, in contact with the skin with a porous gauze dressing and nonirritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance.
(iii) Inhalation studies. (A) The animals should be tested with inhalation equipment designed to sustain a dynamic air flow of 12 to 15 air changes per hour, to ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. As a general rule, to ensure stability of a chamber atmosphere, the total “volume” of the test animals should not exceed 5 percent of the volume of the test chamber. Alternatively, oro-nasal, head only, or whole body individual chamber exposure may be used.
(B) The temperature at which the test is performed should be maintained at 22 °C (±2°). Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(C) Feed and water should be withheld during each daily 6-hour exposure period.
(D) A dynamic inhalation system with a suitable analytical concentration control system should be used. The rate of air flow should be adjusted to ensure that conditions throughout the equipment are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas.
(7) Observation of animals. (i) Each animal should be handled and its physical condition appraised at least once each day.
(ii) Additional observations should be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Clinical signs and mortality should be recorded for all animals. Special attention should be paid to tumor development. The time of onset, location, dimensions, appearance and progression of each grossly visible or palpable tumor should be recorded.
(iv) Body weights should be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter, unless signs of clinical toxicity suggest more frequent weighings to facilitate monitoring of health status.
(v) When the test substance is administered in the feed or drinking water, measurements of feed or water consumption, respectively, should be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise.
(vi) At the end of the study period, all survivors are sacrificed. Moribund animals should be removed and sacrificed when noticed.
(8) Physical measurements. For inhalation studies, measurements or monitoring should be made of the following:
(i) The rate of airflow should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the test substance should be held as constant as practicable, monitored continuously and recorded at least three times during the test period: At the beginning, at an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size analysis should be performed to establish the stability of aerosol concentrations. During exposure, analyses should be conducted as often as necessary to determine the consistency of particle size distribution and homogeneity of the exposure stream.
(iv) Temperature and humidity should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. (i) The following examinations should be made on at least 20 rodents of each sex per dose level:
(A) Certain hematology determinations (e.g., hemoglobin content, packed cell volume, total red blood cells, total white blood cells, platelets, or other measures of clotting potential) should be performed at termination and should be performed at 3 months, 6 months and at approximately 6-month intervals thereafter (for those groups on test for longer than 12 months) on blood samples collected from 20 rodents per sex of all groups. These collections should be from the same animals at each interval. If clinical observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals should be performed. A differential blood count should be performed on samples from animals in the highest dosage group and the controls. Differential blood counts should be performed for the next lower group(s) if there is a major discrepancy between the highest group and the controls. If hematological effects were noted in the subchronic test, hematological testing should be performed at 3, 6, 12, 18 and 24 months for a year study.
(B) Certain clinical biochemistry determinations on blood should be carried out at least three times during the test period: Just prior to initiation of dosing (baseline data), near the middle and at the end of the test period. Blood samples should be drawn for clinical measurements from at least ten rodents per sex of all groups; if possible, from the same rodents at each time interval. Test areas which are considered appropriate to all studies: electrolyte balance, carbohydrate metabolism and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity. Suggested chemical determinations: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, blood urea nitrogen, albumen, creatinine phosphokinase, total cholesterol, total bilirubin and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methemoglobin and cholinesterase activity. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects.
(ii) The following should be performed on at least 10 rodents of each sex per dose level:
(A) Urine samples from the same rodents at the same intervals as hematological examination above, should be collected for analysis. The following determinations should be made from either individual animals or on a pooled sample/sex/group for rodents: appearance (volume and specific gravity), protein, glucose, ketones, bilirubin, occult blood (semi-quantitatively) and microscopy of sediment (semi-quantitatively).
(B) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study. If changes in the eyes are detected, all animals should be examined.
(10) Gross necropsy. (i) A complete gross examination should be performed on all animals, including those which died during the experiment or were killed in moribund conditions.
(ii) The liver, kidneys, adrenals, brain and gonads should be weighed wet, as soon as possible after dissection to avoid drying. For these organs, at least 10 rodents per sex per group should be weighed.
(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination: All gross lesions and tumors; brain-including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; sternum and/or femur with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); female mammary gland; aorta; gall bladder (if present); skin; musculature; peripheral nerve; spinal cord at three levels – cervical, midthoracic, and lumbar; and eyes. In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx and paranasal sinuses should be examined and preserved. In dermal studies, skin from sites of skin painting should be examined and preserved.
(iv) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is considered essential for appropriate and valid histopathological examination.
(v) If other clinical examinations are carried out, the information obtained from these procedures should be available before microscopic examination, since they may provide significant guidance to the pathologist.
(11) Histopathology. (i) The following histopathology should be performed:
(A) Full histopathology on the organs and tissues, listed above, of all non-rodents, of all rodents in the control and high dose groups and of all rodents that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents should be made for evidence of infection since this provides an assessment of the state of health of the animals.
(ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level should be examined for complete histopathology.
(iii) In case the results of the experiment give evidence of substantial alteration of the animals’ normal longevity or the induction of effects that might affect a toxic response, the next lower dose level should be examined as described above.
(iv) An attempt should be made to correlate gross observations with microscopic findings.
(c) Data and reporting – (1) Treatment of results. (i) Data should be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of study results. (i) The findings of a combined chronic toxicity/oncogenicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence, incidence and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailablity of the test substance should be considered.
(iii) In order for a negative test to be acceptable, it should meet the following criteria: No more than 10 percent of any group is lost due to autolysis, cannibalism, or management problems; and survival in each group is no less than 50 percent at 18 months for mice and hamsters and at 24 months for rats.
(3) Test report. (i) In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information should be reported:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(1) Number of animals dying.
(2) Number of animals showing signs of toxicity.
(3) Number of animals exposed.
(B) Individual animal data. (1) Time of death during the study or whether animals survived to termination.
(2) Time of observation of each abnormal sign and its subsequent course.
(3) Body weight data.
(4) Feed and water consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
(6) Hematological tests employed and all results.
(7) Clinical biochemistry tests employed and all results.
(8) Necropsy findings.
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of results where appropriate.
(11) Historical control data, if taken into account.
(ii) In addition, for inhalation studies the following should be reported:
(A) Test conditions. (1) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
(B) Exposure data. These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and should include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
(4) Actual concentration in test breathing zone.
(5) Particle size distribution (e.g. median aerodynamic diameter of particles with standard deviation from the mean).
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) Benitz, K.F. “Measurement of Chronic Toxicity,” Methods of Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications, 1970) pp. 82-131.
(2) D’Aguanno, W. “Drug Safety Evaluation – Pre-Clinical Considerations,” “Industrial Pharmacology: Neuroleptics. Vol. I Ed. S. Fielding and H. Lal. (Mt. Kisco, New York: Futura Publishing Co., 1974) pp. 317-332.
(3) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and Welfare. (Canada: Department of Health and Welfare, 1975).
(4) Fitzhugh, O.G. “Chronic Oral Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975). pp. 36-45.
(5) Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation: Panel on Carcinogenesis. “Report on Cancer Testing in the Safety of Food Additives and Pesticides,” Toxicology and Applied Pharmacology. 20:419-438 (1971).
(6) Goldenthal, E.I., and D’Aguanno, W. “Evaluation of Drugs,” Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd printing 1975) pp.60-67.
(7) International Union Against Cancer. “Carcinogenicity Testing,” IUCC Technical Report Series Vol. 2, Ed. I. Berenblum. (Geneva: International Union Against Cancer, 1969).
(8) Leong, B.K.J., and Laskin, S. “Number and Species of Experimental Animals for Inhalation Carcinogenicity Studies,” Paper presented at Conference on Target Organ Toxicity. September, 1975, Cincinnati, Ohio.
(9) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” A report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(10) National Cancer Institute. Report of the Subtask Group on Carcinogen Testing to the Interagency Collaborative Group on Environmental Carcinogenesis. (Bethesda: United States National Cancer Institute, 1976).
(11) National Center for Toxicological. Report of Chronic Studies Task Force Research Committee. “Appendix B, (Rockville: National Center for Toxicological Research, 1972)).
(12) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal Environmental Pathology and Toxicology. 1:161-182 (1977).
(13) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,” Advances in Modern Toxicology Volume 3, Ed. Kraybill and Mehlman. (Washington, D.C.: Hemisphere Publishing Corp., 1977) pp. 87-171.
(14) Schwartz, E. 1974. “Toxicology of Neuroleptic Agents,” Industrial Pharmacology: Neuroleptics. Ed. S. Fielding and H. Lal. (Mt. Kisco, New York: Futura Publishing Co, 1974) pp. 203-221.
(15) Sontag, J.M., Page, N.P., and Saffiotti, U. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program, 1976).
(16) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (1977).
(17) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,” WHO Technical Report Series No. 426. (Geneva: World Health Organization, 1969).
(18) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,” WHO Technical Report Series No. 563. (Geneva: World Health Organization, 1975).
(19) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
(20) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,” WHO Technical Report Series No. 341. (Geneva: World Health Organization, 1966).
Subpart E – Specific Organ/Tissue Toxicity
§ 798.4100 Dermal sensitization.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a substance, determination of its potential to provoke skin sensitization reactions is important. Information derived from tests for skin sensitization serves to identify the possible hazard to a population repeatedly exposed to a test substance. While the desirability of skin sensitization testing is recognized, there are some real differences of opinion about the best method to use. The test selected should be a reliable screening procedure which should not fail to identify substances with significant allergenic potential, while at the same time avoiding false negative results.
(b) Definitions. (1) Skin sensitization (allergic contact dermatitis) is an immunologically mediated cutaneous reaction to a substance. In the human, the responses may be characterized by pruritis, erythema, edema, papules, vesicles, bullae, or a combination of these. In other species the reactions may differ and only erythema and edema may be seen.
(2) Induction period is a period of at least 1 week following a sensitization exposure during which a hypersensitive state is developed.
(3) Induction exposure is an experimental exposure of a subject to a test substance with the intention of inducing a hypersensitive state.
(4) Challenge exposure is an experimental exposure of a previously treated subject to a test substance following an induction period, to determine whether the subject will react in a hypersensitive manner.
(c) Principle of the test method. Following initial exposure(s) to a test substance, the animals are subsequently subjected, after a period of not less than 1 week, to a challenge exposure with the test substance to establish whether a hypersensitive state has been induced. Sensitization is determined by examining the reaction to the challenge exposure and comparing this reaction to that of the initial induction exposure.
(d) Test procedures. (1) Any of the following seven test methods is considered to be acceptable. It is realized, however, that the methods differ in their probability and degree of reaction to sensitizing substances.
(i) Freund’s complete adjuvant test.
(ii) Guinea-pig maximization test.
(iii) Split adjuvant technique.
(iv) Buehler test.
(v) Open epicutaneous test.
(vi) Mauer optimization test.
(vii) Footpad technique in guinea pig.
(2) Removal of hair is by clipping, shaving, or possibly by depilation, depending on the test method used.
(3) Animal selection – (i) Species and strain. The young adult guinea pig is the preferred species. Commonly used laboratory strains should be employed. If other species are used, the tester should provide justification/reasoning for their selection.
(ii) Number and sex. (A) The number and sex of animals used will depend on the method employed.
(B) The females should be nulliparous and nonpregnant.
(4) Control animals. (i) Periodic use of a positive control substance with an acceptable level of reliability for the test system selected is recommended;
(ii) Animals may act as their own controls or groups of induced animals can be compared to groups which have received only a challenge exposure.
(5) Dose levels. The dose level will depend upon the method selected.
(6) Observation of animals. (i) Skin reactions should be graded and recorded after the challenge exposures at the time specified by the methodology selected. This is usually at 24, 48, and 72, hours. Additional notations should be made as necessary to fully describe unusual responses;
(ii) Regardless of method selected, initial and terminal body weights should be recorded.
(7) Procedures. The procedures to be used are those described by the methodology chosen.
(e) Data and reporting. (1) Data should be summarized in tabular form, showing for each individual animal the skin reaction, results of the induction exposure(s) and the challenge exposure(s) at times indicated by the method chosen. As a minimum, the erythema and edema should be graded and any unusual finding should be recorded.
(2) Evaluation of the results. The evaluation of results will provide information on the proportion of each group that became sensitized and the extent (slight, moderate, severe) of the sensitization reaction in each individual animal.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information should be reported:
(i) A description of the method used and the commonly accepted name.
(ii) Information on the positive control study, including positive control used, method used, and time conducted.
(iii) The number and sex of the test animals.
(iv) Species and strain.
(v) Individual weights of the animals at the start of the test and at the conclusion of the test.
(vi) A brief description of the grading system.
(vii) Each reading made on each individual animal.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Buehler, E.V. “Delayed Contact Hypersensitivity in the Guinea Pig,” Archives Dermatology. 91:171 (1965).
(2) Draize, J.H. “Dermal Toxicity,” Food Drug Cosmetic Law Journal. 10:722-732 (1955).
(3) Klecak, G. “Identification of Contact Allergens: Predictive Tests in Animals,” Advances in Modern Toxicology: Dermatology and Pharmacology. Ed. F.N. Marzulli and H.I. Maibach. (Washington, D.C.: Hemisphere Publishing Corp., 1977) 4:305-339).
(4) Klecak, G., Geleick, H., Grey, J.R. “Screening of Fragrance Materials for Allergenicity in the Guinea Pig.-1. Comparison of Four Testing Methods,” Journal of the Society of Cosmetic Chemists. 28:53-64 (1977).
(5) Magnusson, B., Kligman, A.M. “The Identification of Contact Allergens by Animal Assay,” The Guinea Pig Maximization Test. The Journal of Investigative Dermatology. 52:268-276 (1973).
(6) Maguire, H.C. “The Bioassay of Contact Allergens in the Guinea Pig” Journal of the Society of Cosmetic Chemists. 24:151-162 (1973).
(7) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. “The Optimization Test in the Guinea Pig. A Method for the Predictive Evaluation of the Contact Allergenicity of Chemicals,” Agents and Actions. (Basel: Birkhauser Verlag, 1975) Vol. 5/2.
(8) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. “The Optimization Test in the Guinea Pig: A Method for the Predictive Evaluation of the Contact Allergenicity of Chemicals,” International Congress Series Excerpta Medica No. 376, (1975) Vol. 203.
§ 798.4350 Inhalation developmental toxicity study.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of an inhalable material such as a gas, volatile substance, or aerosol/particulate, determination of the potential developmental toxicity is important. The inhalation developmental toxicity study is designed to provide information on the potential hazard to the unborn which may arise from exposure of the mother during pregnancy.
(b) Definitions. (1) Developmental toxicity is the property of a chemical that causes in utero death, structural or functional abnormalities or growth retardation during the period of development.
(2) “Aerodynamic diameter” applies to the behavioral size of particles of aerosols. It is the diameter of a sphere of unit density which behaves aerodynamically like the particles of the test substance. It is used to compare particles of different sizes, shapes, and densities and to predict where in the respiratory tract such particles may be deposited. This term is used in contrast to “optical,” “measured” or “geometric” diameters which are representation of actual diameters which in themselves cannot be related to deposition within the respiratory tract.
(3) “Geometric mean diameter” or “median diameter” is the calculated aerodynamic diameter which divides the particles of an aerosol in half based on the weight of the particles. Fifty percent of the particles by weight will be larger than the median diameter and 50 percent of the particles will be smaller than the median diameter. The median diameter and its geometeric standard deviation are used to statistically describe the particle size distribution of any aerosol based on the weight and size of the particles.
(4) “Inhalable diameter” refers to that aerodynamic diameter of a particle which is considered to be inhalable for the organism. It is used to refer to particles which are capable of being inhaled and may be deposited anywhere within the respiratory tract from the trachea to the deep lung (the alveoli). For man, the inhalable diameter is considered here as 15 micrometers or less.
(5) “Concentration” refers to an exposure level. Exposure is expressed as weight or volume of test substance per volume of air (mg/1), or as parts per million (ppm).
(6) “No-observed-effect level” is the maximum concentration in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight or volume of test substance given daily per unit volume of air.
(c) Principle of the test method. The test substance is administered in graduated concentrations, for at least that part of the pregnancy covering the major period of organogenesis, to several groups of pregnant experimental animals, one exposure level being used per group. Shortly before the expected date of delivery, the pregnant females are sacrificed, the uteri removed, and the contents examined for embryonic or fetal deaths, and live fetuses.
(d) Limit test. If a test at an exposure of 5 mg/1 (actual concentration of respirable substances) or, where this is not possible due to physical or chemical properties of the test substance, the maximum attainable concentration, produces no observable developmental toxicity, then a full study using three exposure levels might not be necessary.
(e) Test procedures – (1) Animal selection – (i) Species and strain. Testing shall be performed in at least two mamalian species. Commonly used species include the rat, mouse, rabbit, and hamster. If other mamalian species are used, the tester shall provide justification/reasoning for their selection. Commonly used laboratory strains shall be employed. The strain shall not have low fecundity and shall preferably be characterized for its sensitivity to developmental toxins.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant female animals shall be used at each exposure level.
(iv) Number of animals. At least 20 pregnant rats, mice, or hamsters or 12 pregnant rabbits are required at each exposure level. The objective is to ensure that sufficient pups are produced to permit meaningful evaluation of the potential developmental toxicity of the test substance.
(2) Control group. A concurrent control group shall be used. This group shall be exposed to clean, filtered air under conditions identical to those used for the group exposed to the substance of interest. In addition, a vehicle-exposed group may be necessary when the substance under study requires a vehicle for delivery. It is recommended that during preliminary range finding studies, air vs. vehicle exposure be compared. If there is no substantial difference, air exposure itself would be an appropriate control. If vehicle and air exposure yield different results, both vehicle and air exposed control groups are recommended.
(3) Concentration levels and concentration selection. (i) At least three concentration levels with a control and, where appropriate, a vehicle control, shall be used.
(ii) The vehicle shall neither be developmentally toxic nor have effects on reproduction.
(iii) To select the appropriate concentration levels, a pilot or trial study may be advisable. Since pregnant animals have an increased minute ventilation as compared to non-pregnant animals, it is recommended that the trial study be conducted in pregnant animals. Similarly, since presumably the minute ventilation will vary with progression of pregnancy, the animals should be exposed during the same period of gestation as in the main study. In the trial study, the concentration producing embryonic or fetal lethalities or maternal toxicity should be determined.
(iv) Unless limited by the physical/chemical nature or biological properties of the substance, the highest concentration level shall induce some overt maternal toxicity such as reduced body weight or body weight gain, but not more than 10 percent maternal deaths.
(v) The lowest concentration level should not produce any grossly observable evidence of either maternal or developmental toxicity.
(vi) Ideally, the intermediate concentration level(s) shall produce minimal observable toxic effects. If more than one intermediate concentration is used, the concentration levels shall be spaced to produce a gradation of toxic effects.
(4) Exposure duration. The duration of exposure shall be at least six hours daily allowing appropriate additional time for chamber equilibrium.
(5) Observation period. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The exposure period shall cover the period of major organogenesis. This may be taken as days 6 to 15 for rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
(6) Inhalation exposure. (i)(A) The animals shall be tested in inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour and ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber.
(B) Pregnant animals shall not be subjected to beyond the minimum amount of stress. Since whole-body exposure appears to be the least stressful mode of exposure, it is the method preferred. In general oro-nasal or head-only exposure, which is sometimes used to avoid concurrent exposure by the dermal or oral routes, is not recommended because of the associated stress accompanying the restraining of the animals. However, there may be specific instances where it may be more appropriate than whole-body exposure. The tester shall provide justification/reasoning for its selection.
(ii) A dynamic inhalation system with a suitable flow control system shall be used. The rate of air flow shall be adjusted to ensure that conditions throughout the exposure chamber are essentially the same. Test material distribution should be established before animals are committed to dosing. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas.
(iii) The temperature at which the test is performed should be maintained at 22 °C (±2°) for rodents or 20 °C (±3°) for rabbits. Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(7) Physical measurements. Measurements or monitoring should be made of the following:
(i) The rate of airflow shall be monitored continuously but shall be recorded at least every 30 minutes.
(ii) The actual concentration of the test substance shall be measured in the breathing zone. During the exposure period the actual concentrations of the test substance shall be held as constant as practicable, monitored continously or intermittently depending on the method of analysis and measured at least at the beginning, at an intermediate time and at the end of the exposure period.
(iii) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. During exposure, analysis shall be conducted as often as necessary to determine the consistency of particle size distribution.
(iv) Temperature and humidity shall be monitored continuously and be recorded at least every 30 minutes.
(8) Food and water during exposure period. Food should be withheld during exposure. Water may or may not be withheld. If it is not withheld it should not come in direct contact with the test atmospheres.
(9) Observation of animals. (i) A gross examination shall be made at least once each day.
(ii) Additional observations should be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree and duration.
(iv) Cage-side observations shall include, but not be limited to: Changes in skin and fur, eye and mucous membranes, as well as respiratory, autonomic and central nervous systems, somatomotor activity and behavioral pattern. Particular attention should be directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma.
(v) Measurements should be made weekly of food consumption for all animals in the study.
(vi) Animals shall be weighed at least weekly.
(vii) Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination.
(10) Gross necropsy. (i) At the time of sacrifice or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
(ii) Immediately after sacrifice or death, the uterus shall be removed, weighed, and the contents examined for embryonic or fetal deaths and the number of viable fetuses. Gravid uterine weights should not be obtained from dead animals if autolysis or where decomposition has occurred. The degree of resorption shall be described in order to help estimate the relative time of death.
(iii) The number of corpora lutea shall be determined for all species except mice.
(iv) The sex of the fetuses shall be determined and they shall be weighed individually, the weights recorded, and the mean fetal weight derived.
(v) Following removal, each fetus shall be examined externally.
(vi) For rats, mice and hamsters, one-third to one-half of each litter shall be prepared and examined for skeletal anomalies, and the remaining part of each litter shall be prepared and examined for soft tissue anomalies using appropriate methods.
(vii) For rabbits, each fetus shall be examined by careful dissection for visceral anomalies and then examined for skeletal anomalies.
(f) Data and reporting – (1) Treatment of results. Data shall be summarized in tabular form, showing for each test group: the number of animals at the start of the test, the number of pregnant animals, the number and percentages of live fetuses and the number of fetuses with any soft tissue or skeletal abnormalities.
(2) Evaluation of results. The findings of a developmental toxicity study shall be evaluated in terms of the observed effects and the exposure levels producing effects. It is necessary to consider the historical developmental toxicity data on the species/strain tested. A properly conducted developmental toxicity study should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, methods of conditioning air, and the method of housing the animals in a test chamber when this apparatus is used.
(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and shall include:
(A) Airflow rates through the inhalation equipment.
(B) Temperature of air.
(C) Nominal concentration – total amount of test substance fed into the inhalation equipment divided by volume of air (no standard deviation).
(D) Measured total concentrations (particulate and/or gaseous phases) in test breathing zone.
(E) Particle size distribution (e.g., median aerodynamic diameter of particles with geometric standard deviation) including estimates of the percents of inhalable and non-inhalable portions for the test animals.
(iii) Animal data. (A) Toxic response data by concentration.
(B) Species and strain.
(C) Date of death during the study or whether animals survived to termination.
(D) Date of onset and duration of each abnormal sign and its subsequent course.
(E) Feed, body weight and uterine weight data.
(F) Pregnancy and litter data.
(G) Fetal data (live/dead, sex, soft tissue and sketetal defects, resorptions).
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity. Minister of Health and Welfare (Canada: Department of Health and Welfare, 1975).
(2) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances.” A report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(3) World Health Organization. Principles for the Testing of Drugs for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World Health Organization, 1967).
§ 798.4700 Reproduction and fertility effects.
(a) Purpose. This guideline for two-generation reproduction testing is designed to provide general information concerning the effects of a test substance on gonadal function, conception, parturition, and the growth and development of the offspring. The study may also provide information about the effects of the test substance on neonatal morbidity, mortality, and preliminary data on teratogenesis and serve as a guide for subsequent tests.
(b) Principle of the test method. The test substance is administered to parental (P) animals prior to their mating, during the resultant pregnancies, and through the weaning of their F
(c) Test procedures – (1) Animal selection – (i) Species and strain. The rat is the preferred species. If another mammalian species is used, the tester shall provide justification/reasoning for its selection. Strains with low fecundity shall not be used.
(ii) Age. Parental (P) animals shall be about 5 to 8 weeks old at the start of dosing.
(iii) Sex. (A) For an adequate assessment of fertility, both males and females shall be studied.
(B) The females shall be nulliparous and non-pregnant.
(iv) Number of animals. Each test and control group shall contain at least 20 males and a sufficient number of females to yield at least 20 pregnant females at or near term.
(2) Control groups. (i) A concurrent control group shall be used. This group shall be an untreated or sham treated control group or if a vehicle is used in administering the test substance, a vehicle control group.
(ii) If a vehicle is used in administering the test substance, the control group shall receive the vehicle in the highest volume used.
(iii) If a vehicle or other additive is used to facilitate dosing, it shall not interfere significantly with absorption of the test substance or produce toxic effects.
(3) Dose levels and dose selection. (i) At least three dose levels and a concurrent control shall be used.
(ii) The highest dose level should induce toxicity but not high levels of mortality in the parental (P) animals.
(iii) The lowest dose level should not produce any grossly observable evidence of toxicity.
(iv) Ideally the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, dose levels should be spaced to produce a gradation of toxic effects.
(4) Exposure conditions. The animals should be dosed with the test substance, ideally, on a 7 days per week basis.
(i) Dosing, mating, delivery, and sacrifice schedule.
(A) Daily dosing of the parental (P) males and females shall begin when they are 5 to 8 weeks old. For both sexes, dosing shall be continued for at least 10 weeks before the mating period.
(B) Dosing of P males shall continue through the 3 week mating period. At the end of the mating period, P males may be sacrificed and examined, or may be retained for possible production of a second litter. If these animals are retained for a second litter, dosing shall be continued. Dosing of the F
(C) Daily dosing of the P females shall continue through the three week mating period, pregnancy, and to the weaning of the F
(ii) All animals are sacrificed as scheduled.
(A) All P males should be sacrificed at the end of the 3-week mating period, or may be retained for possible production of a second litter. If these animals are retained for a second litter, dosing shall be continued.
(B) F
(C) F
(D) The P females should be sacrificed upon weaning of their F
(E) F
(5) Administration of the test substance – (i) Oral studies. (A) It is recommended that the test substance be administered in the diet or drinking water.
(B) If administered by gavage or capsule, the dosage administered to each animal prior to mating shall be based on the individual animal’s body weight and adjusted weekly. During pregnancy the dosage shall be based on the body weight at day 0 and 6 of pregnancy.
(ii) If another route of administration is used, the tester should provide justification and reasoning for its selection.
(6) Mating procedure – (i) Parental. (A) For each mating, each female shall be placed with a single male from the same dose level until pregnancy occurs or 1 week has elapsed. If mating has not occurred after 1 week, the female shall be placed with a different male. Paired matings should be clearly identified.
(B) Those pairs that fail to mate should be evaluated to determine the cause of the apparent infertility. This may involve such procedures as additional opportunities to mate with proven fertile males or females, histological examination of the reproductive organs, and examination of the estrus or spermatogenic cycles.
(C) Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day vaginal plugs or sperm are found.
(ii) F
(B) F
(iii) Special housing. After evidence of copulation, pregnant animals shall be caged separately in delivery or maternity cages. Pregnant animals shall be provided with nesting materials when parturition is near.
(iv) Standardization of litter sizes. (A) On day 4 after birth, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter.
(B) Whenever the number of male or female pups prevents having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) is permitted. Adjustments are not appropriate for litters of less than 8 pups.
(C) Elimination of runts only is not appropriate.
(D) Adjustments of the F
(7) Observation of animals. (i) A gross examination shall be made at least once each day. Pertinent behavioral changes, signs of difficult or prolonged parturition, and all signs of toxicity, including mortality, shall be recorded. These observations shall be reported for each individual animal. Food consumption for all animals shall be monitored weekly except during the mating period.
(ii) The duration of gestation shall be calculated from day 0 of pregnancy.
(iii) Each litter should be examined as soon as possible after delivery for the number of pups, stillbirths, live births, sex, and the presence of gross anomalies. Live pups should be counted and litters weighed at birth or soon thereafter, and on days 4, 7, 14, and 21 after parturition.
(iv) Physical or behavioral abnormalities observed in the dams of offspring shall be recorded.
(v) P males and females shall be weighed on the first day of dosing and weekly thereafter. F
(8) Gross necropsy. (i) A complete gross examination shall be performed on all adult animals, including those which died during the experiment or were killed in moribund conditions.
(ii) Special attention shall be directed to the organs of the reproductive system.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: Vagina; uterus; ovaries; testes; epididymides; seminal vesicles; prostate, pituitary gland; and, target organ(s) when previously identified of all P and F
(9) Histopathology. Except if carried out in other studies of comparable duration and dose levels the following histopathology shall be performed:
(i) Full histopathology on the organs listed above for all high dose, and control P
(ii) Organs demonstrating pathology in these animals shall then be examined in animals from the other dose groups.
(iii) Microscopic examination shall be made of all tissues showing gross pathological changes.
(d) Data and reporting – (1) Treatment of results. Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals pregnant, the types of change and the percentage of animals displaying each type of change.
(2) Evaluation of study results. (i) An evaluation of test results, including the statistical analysis, based on the clinical findings, the gross necropsy findings, and the microscopic results shall be made and supplied. This should include an evaluation of the relationship, or lack thereof, between the animals’ exposure to the test substance and the incidence and severity of all abnormalities.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Toxic response data by sex and dose, including fertility, gestation, viability and lactation indices, and length of gestation.
(ii) Species and strain.
(iii) Date of death during the study or whether animals survived to termination.
(iv) Toxic or other effects on reproduction, offspring, or postnatal growth.
(v) Date of observation of each abnormal sign and its subsequent course.
(vi) Body weight data for P, F
(vii) Necropsy findings.
(viii) Detailed description of all histopathological findings.
(ix) Statistical treatment of results where appropriate.
(e) References. For additional background information on this test guideline the following references should be consulted:
(1) Clermont, Y., Perry, B. “Quantitative Study of the Cell Population of the Seminiferous Tubules in Immature Rats,” American Journal of Anatomy. 100:241-267 (1957).
(2) Goldenthal, E.I. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use. Drug Review Branch, Division of Toxicological Evaluation, Bureau of Science, Food and Drug Administration, Washington, DC (1966).
(3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W. Fertility and Sterility. (New York: American Elsevier Publishing Co., Inc., 1973).
(4) Oakberg, E.F. “Duration of Spermatogenesis in the Mouse and Timing of Stages of the Cycle of the Seminiferous Epithelium,” American Journal of Anatomy. 9:507-516 (1956).
(5) Roosen-Runge, E.C. “The Process of Spermatogenesis in Mammals,” Biological Review. 37:343-377 (1962).
§ 798.4900 Developmental toxicity study.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, determination of the potential developmental toxicity is important. The developmental toxicity study is designed to provide information on the potential hazard to the unborn which may arise from exposure of the mother during pregnancy.
(b) Definitions. (1) Developmental toxicity is the property of a chemical that causes in utero death, structural or functional abnormalities or growth retardation during the period of development.
(2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of a test animal (e.g., mg/kg).
(3) No-observed-effect level is the maximum concentration in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight of test substance given daily per unit weight of test animal (mg/kg)
(c) Principle of the test method. The test substance is administered in graduated doses for at least part of the pregnancy covering the major period of organogenesis, to several groups of pregnant experimental animals, one dose level being used per group. Shortly before the expected date of delivery, the pregnant females are sacrificed, the uteri removed, and the contents examined for embryonic or fetal deaths, and live fetuses.
(d) Limit test. If a test at an exposure of at least 1000 mg/kg body weight, using the procedures described for this study, produces no observable developmental toxicity, then a full study using three dose levels might not be necessary.
(e) Test procedures – (1) Animal selection – (i) Species and strain. Testing shall be performed in at least 2 mammalian species. Commonly used species include the rat, mouse, rabbit, and hamster. If other mammalian species are used, the tester shall provide justification/reasoning for their selection. Commonly used laboratory strains shall be employed. The strain shall not have low fecundity and shall preferably be characterized for its sensitivity to developmental toxins.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant female animals shall be used at each dose level.
(iv) Number of animals. At least 20 pregnant rats, mice or hamsters or 12 pregnant rabbits are required at each dose level. The objective is to ensure that sufficient pups are produced to permit meaningful evaluation of the potential developmental toxicity of the test substance.
(2) Control group. A concurrent control group shall be used. This group shall be an untreated or sham treated control group, or, if a vehicle is used in administering the test substance, a vehicle control group. Except for treatment with the test substance, animals in the control group(s) shall be handled in an identical manner to test group animals.
(3) Dose levels and dose selection. (i) At least 3 dose levels with a control and, where appropriate, a vehicle control, shall be used.
(ii) The vehicle shall neither be developmentally toxic nor have effects on reproduction.
(iii) To select the appropriate dose levels, a pilot or trial study may be advisable. It is not always necessary to carry out a trial study in pregnant animals. Comparison of the results from a trial study in non-pregnant, and the main study in pregnant animals will demonstrate if the test substance is more toxic in pregnant animals. If a trial study is carried out in pregnant animals, the dose producing embryonic or fetal lethalities or maternal toxicity shall be determined.
(iv) Unless limited by the physical/chemical nature or biological properties of the substance, the highest dose level shall induce some overt maternal toxicity such as reduced body weight or body weight gain, but not more than 10 percent maternal deaths.
(v) The lowest dose level should not produce any grossly observable evidence of either maternal or developmental toxicity.
(vi) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate concentration is used, the concentration levels should be spaced to produce a gradation of toxic effects.
(4) Observation period. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The dose period shall cover the period of major organogenesis. This may be taken as days 6 to 15 for rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
(5) Administration of test substance. The test substance or vehicle is usually administered orally, by oral intubation unless the chemical or physical characteristics of the test substance or pattern of human exposure suggest a more appropriate route of administration. The test substance shall be administered approximately the same time each day.
(6) Exposure conditions. The female test animals are treated with the test substance daily throughout the appropriate treatment period. When given by gavage, the dose may be based on the weight of the females at the start of substance administration, or, alternatively, in view of the rapid weight gain which takes place during pregnancy, the animals may be weighed periodically and the dosage based on the most recent weight determination.
(7) Observation of animals. (i) A gross examination shall be made at least once each day.
(ii) Additional observations shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree and duration.
(iv) Cage-side observations shall include, but not be limited to: changes in skin and fur, eye and mucous membranes, as well as respiratory, autonomic and central nervous systems, somatomotor activity and behavioral pattern.
(v) Measurements should be made weekly of food consumption for all animals in the study.
(vi) Animals shall be weighed at least weekly.
(vii) Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination.
(8) Gross necropsy. (i) At the time of sacrifice or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
(ii) Immediately after sacrifice or as soon as possible after death, the uterus shall be removed and the contents examined for embryonic or fetal deaths and the number of viable fetuses. The degree of resorption shall be described in order to help estimate the relative time of death of the conceptus. The weight of the gravid uterus should be recorded for dams that are sacrificed. Gravid uterine weights should not be obtained from dead animals if autolysis or decomposition has occurred.
(iii) The number of corpora lutea shall be determined for all species except mice.
(iv) The sex of the fetuses shall be determined and they shall be weighed individually, the weights recorded, and the mean fetal weight derived.
(v) Following removal, each fetus shall be examined externally.
(vi) For rats, mice and hamsters, one-third to one-half of each litter shall be prepared and examined for skeletal anomalies, and the remaining part of each litter shall be prepared and examined for soft tissue anomalies using appropriate methods.
(vii) For rabbits, each fetus shall be examined by careful dissection for visceral anomalies and then examined for skeletal anomalies.
(f) Data and reporting – (1) Treatment of results. Data shall be summarized in tablular form, showing for each test group: the number of animals at the start of the test, the number of pregnant animals, the number and percentages of live fetuses and the number of fetuses with any soft tissue or skeletal abnormalities.
(2) Evaluation of results. The findings of a developmental toxicity study shall be evaluated in terms of the observed effects and the exposure levels producing effects. It is necessary to consider the historical developmental toxicity data on the species/strain tested. A properly conducted developmental toxicity study should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Toxic response data by concentration.
(ii) Species and strain.
(iii) Date of death during the study or whether animals survived to termination.
(iv) Date of onset and duration of each abnormal sign and its subsequent course.
(v) Food, body weight and uterine weight data.
(vi) Pregnancy and litter data.
(vii) Fetal data (live/dead, sex, soft tissue and skeletal defects, resorptions).
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, mutagenicity and Teratogenicity. Minister of Health and Welfare (Canada: Department of Health and Welfare, 1975).
(2) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances.” A report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(3) World Health Organization. Principles for the Testing of Drugs for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World Health Organization, (1967).
Subpart F – Genetic Toxicity
§ 798.5195 Mouse biochemical specific locus test.
(a) Purpose. The mouse biochemical specific locus test (MBSL) may be used to detect and quantitate mutations originating in the germ line of a mammalian species.
(b) Definitions. (1) A biochemical specific locus mutation is a genetic change resulting from a DNA lesion causing alterations in proteins that can be detected by electrophoretic methods.
(2) The germ line is comprised of the cells in the gonads of higher eukaryotes, which are the carriers of the genetic information for the species.
(c) Reference substances. Not applicable.
(d) Test method – (1) Principle. The principle of the MBSL is that heritable damage to the genome can be detected by electrophoretic analysis of proteins in the tissues of the progeny of mice treated with germ cell mutagens.
(2) Description. For technical reasons, males rather than females are generally treated with the test chemical. Treated males are then mated to untreated females to produce F1 progeny. Both blood and kidney samples are taken from progeny for electrophoretic analysis. Up to 33 loci can be examined by starch-gel electrophoresis and broad-range isoelectric focussing. Mutants are identified by variations from the normal electrophoretic pattern. Presumed mutants are bred to confirm the genetic nature of the change.
(3) Animal selection – (i) Species and strain. Mice shall be used as the test species. Although the biochemical specific locus test could be performed in a number of in bred strains, in the most frequently used cross, C57BL/6 females are mated to DBA/2 males to produce (C57BL/6 × DBA/2) F1 progeny for screening.
(ii) Age. Healthy, sexually-mature (at least 8 weeks old) animals shall be used for treatment and breeding.
(iii) Number. A decision on the minimum number of treated animals should take into account possible effects of the test chemical on the fertility of the treated animals. Other considerations should include:
(A) The production of concurrent spontaneous controls.
(B) The use of positive controls.
(C) The power of the test.
(4) Control groups – (i) Concurrent controls. An appropriate number of concurrent control loci shall be analyzed in each experiment. These should be partly derived from matings of untreated animals (from 5 to 20 percent ofthe treated matings), although some data on control loci can be taken from the study of the alleles transmitted from the untreated parent in the experimental cross. However, any laboratory which has had no prior experience with the test shall produce a spontaneous control sample of about 5,000 progeny animals and a positive control (using 100 mg/kg ethylnitrosourea) sample of at least 1,200 offspring.
(ii) Historical controls. Long-term, accumulated spontaneous control data (currently, 1 mutation in 1,200,000 control loci screened) are available for comparative purposes.
(5) Test chemicals – (i) Vehicle. When possible, test chemicals shall be dissolved or suspended in distilled water or buffered isotonic saline. Water-insoluble chemicals shall be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test chemical nor produce major toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. Usually, only one dose need be tested. This should be the maximum tolerated dose (MTD), the highest dose tolerated without toxic effects. Any temporary sterility induced due to elimination of spermatogonia at this dose must be of only moderate duration, as determined by are turn of males to fertility within 80 days after treatment. For evaluation of dose-response, it is recommended that at least two dose levels be tested.
(iii) Route of administration. Acceptable routes of administration include, but are not limited to, gavage, inhalation, and mixture with food or water, and intraperitoneal or intravenous injections.
(e) Test performance – (1) Treatment and mating. Male DBA/2 mice shall be treated with the test chemical and mated to virgin C57BL/6 females immediately after cessation of treatment. Each treated male shall be mated to new virgin C57BL/6 females each week. Each pairing will continue for a week until the next week’s mating is to begin. This mating schedule permits sampling of all post-spermatogonial stages of germ-cell development during the first 7 weeks after exposure. Spermatogonial stem cells are studied thereafter. Repeated mating cycles should be conducted until sufficient offspring have been obtained to meet the power criterion of the assay for spermatogonial stem cells.
(2) Examination of offspring – (i) Birth and weaning. Offspring shall be examined at birth and at weaning for externally detectable changes in morphology and behavior; these could be due to dominant mutations. Such characteristics may include, but are not limited to, variations in coat color, appearance of eyes, size (in which case weighing of variant animals and littermates should be carried out), fur texture, etc. Gross changes in external form and behavior shall also be sought. Scrutiny of such visible characteristics of all animals shall be made during all subsequent manipulations of the animals.
(ii) Tissue sampling. Blood (about 0.1 mL) and one kidney shall be removed from progeny mice under anesthesia. Both tissues are then prepared for analysis by electrophoresis.
(iii) Electrophoresis. The gene products of 6 loci shall be analyzed in the blood sample by broad-range isoelectric focussing and of 27 loci in the kidney sample by starch-gel electrophoresis and enzyme-specific staining. Details on these procedures are included in paragraphs (g)(1) through (g)(3) of this section.
(iv) Mutant identification. Presumptive electrophoretic mutants shall be identified by variation from the normal electrophoretic banding patterns. Reruns of all variant samples shall be performed to confirm the presence of altered banding patterns. Samples from parents of progeny exhibiting banding pattern variations shall be assayed to determine whether the variant was induced by the experimental treatment or was pre-existing. All treatment-induced variants are bred to determine the genetic nature of the change.
(f) Data and reports – (1) Treatment of results. Data shall be presented in tabular form and shall permit independent analysis of cell stage-specific effects, and dose-dependent phenomena. The data shall be recorded and analyzed in such a way that clusters of identical mutations are clearly identified. The individual mutants detected shall be thoroughly described. In addition, concurrent positive control data (if employed) and spontaneous control data shall also be tabulated. These concurrent controls shall be added to, as well as compared with, the historical control data.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive response, one of which is a statistically significant dose-related increase in the frequency of electrophoretic mutations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of these test points.
(ii) A test chemical which does not produce a statistically significant increase in the frequency of electrophoretic mutations over the spontaneous frequency, or a statistically significant and reproducible positive response for at least one of the test points, is considered nonmutagenic in this system, provided that the sample size is sufficient to exclude a biologically significant increase in mutation frequency.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the MBSL indicate that, under the test conditions, the test chemical induces heritable gene mutations in a mammalian species.
(ii) Negative results indicate that, under the test conditions, the test chemical does not induce heritable genemutations in a mammalian species.
(5) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, and paragraph (h) of this section, the following specific information shall be reported:
(i) Strain, age and weight of animals used; numbers of animals of each sex in experimental and control groups.
(ii) Test chemical vehicle, doses used, rationale for dose selection, and toxicity data, if available.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Number of loci screened for both treated and spontaneous data.
(vi) Criteria for scoring mutants.
(vii) Number of mutants found/locus.
(viii) Loci at which mutations were found.
(ix) Use of concurrent negative and positive controls.
(x) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline, the following references should be consulted:
(1) Personal communication from Susan E. Lewis, Ph.D. to Dr. Michael Cimino, U.S. EPA, OPPT, October 5, 1989.
(2) Johnson, F.M., G.T. Roberts, R.K. Sharma, F.Chasalow, R. Zweidinger, A. Morgan, R.W. Hendren, and S.E.Lewis. “The detection of mutants in mice by electrophoresis: Results of a model induction experiment with procarbazine.” Genetics 97:113-124 (1981).
(3) Johnson, F.M. and S.E. Lewis. “Mutation rate determinations based on electrophoretic analysis of laboratory mice.” Mutation Research 82:125-135 (1981a).
(4) Johnson, F.M. and S.E. Lewis. “Electrophoretically detected germinal mutations induced by ethylnitrosourea in the mouse.” Proceedings of the National Academy of Sciences 78:3138-93141 (1981b).
(5) Lewis, S.E., C. Felton, L.B. Barnett, W. Generoso, N. Cacheiro, and M.D. Shelby. “Dominant visible and electrophoretically expressed mutations induced in male mice exposed to ethylene oxide by inhalation.” Environmental Mutagenesis 8:867-872 (1986).
(h) Additional requirements. Testing facilities conducting the mouse biochemical specific locus test in accordance with this section shall, in addition to adhering to the provisions of §§ 792.190 and 792.195 of this chapter, obtain, adequately identify, and retain for at least 10 years, acceptable 35-mm photographs (and their negatives) of the stained isoelectric-focussing columns and the stained starch-gels obtained following analyses of blood and kidney preparations, respectively, from mutant mice, their siblings, and their parents.
§ 798.5200 Mouse visible specific locus test.
(a) Purpose. The mouse visible specific locus test (MSLT) may be used to detect and quantitate mutations in the germ line of a mammalian species.
(b) Definitions. (1) A visible specific locus mutation is a genetic change that alters factors responsible for coat color and other visible characteristics of certain mouse strains.
(2) The germ line is the cells in the gonads of higher eukaryotes which are the carriers of the genetic information for the species.
(c) Reference substances. Not applicable.
(d) Test method – (1) Principle. (i) The principle of the MSLT is to cross individuals who differ with respect to the genes present at certain specific loci, so that a genetic alteration involving the standard gene at any one of these loci will produce an offspring detectably different from the standard heterozygote. The genetic change may be detectable by various means, depending on the loci chosen to be marked.
(ii) Three variations of the method currently exist for detecting newly arising point mutations in mouse germ cells:
(A) The visible specific locus test using either 5 or 7 loci.
(B) The biochemical specific locus test using up to 20 enzymes.
(C) The test for mutations at histocompatibility loci.
(iii) Of the three tests, the visible specific locus test has been most widely used in assessing genetic hazard due to environmental agents. It is the method described in this guideline.
(2) Description. For technical reasons, males rather than females are generally treated with the test agent. Treated males are then mated to females which are genetically homozygous for certain specific visible marker loci. Offspring are examined in the next generation for evidence that a new mutation has arisen.
(3) Animal selection – (i) Species and strain. Mice shall be used as the test species. Male mice shall be either (C
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. A decision on the minimum number of treated animals should take into account the spontaneous variation of the biological characterization being evaluated. Other considerations should include:
(A) The use of either historical or concurrent controls.
(B) The power of the test.
(C) The minimal rate of induction required.
(D) The use of positive controls.
(E) The level of significance desired.
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups – (i) Concurrent controls. The use of positive or spontaneous controls is left to the discretion of the investigator. However, any laboratory which has had no prior experience with the test shall, at its first attempt, produce a negative control sample of 20,000 and a positive control, using 100 mg/kg 1-ethyl-nitrosourea, in a sample of 5,000 offspring.
(ii) Historical controls. Long term, accumulated spontaneous control data of 43/801,406 are available for comparative purposes.
(5) Test chemicals – (i) Vehicle. When possible, test chemicals should be dissolved or suspended in distilled water or isotonic saline buffered appropriately, if needed, for stability. Water-insoluble chemicals shall be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test compound nor produce major toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. Usually, only one dose level need be tested. This should be the highest dose tolerated without toxic effects, provided that any temporary sterility induced due to elimination of spermatagonia is of only moderate duration, as determined by a return of males to fertility within 80 days after treatment. For evaluation of dose-response, it is recommended that at least two dose levels be tested.
(iii) Route of administration. Acceptable routes of administration include gavage, inhalation, admixture with food or water, and IP or IV injections.
(e) Test performance – (1) Treatment and mating. Hybrid F
(2) Examination of offspring. (i) Offspring may be examined at (or soon after) birth but must be examined at about 3 weeks of age at which time the numbers of mutant and nonmutant offspring in each litter shall be recorded.
(ii) Nonmutant progeny should be discarded. Mutant progeny shall be subjected to genetic tests for verification.
(f) Data and report – (1) Treatment of results. Data shall be presented in tabular form and shall permit independent analysis of cell stage specific effects and dose dependent phenomena. The data shall be recorded and analyzed in such a way that clusters of identical mutations are clearly identified. The individual mutants detected shall be thoroughly described. In addition, concurrent positive and negative control data, if they are available, shall be tabulated so that it is possible to differentiate between concurrent (when available) and long-term accumulated mutation frequencies.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of specific locus mutations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of specific locus mutations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the MSLT indicate that under the test conditions the test substance induces heritable gene mutations in the test species.
(ii) Negative results indicate that under the test conditions the test substance does not induce heritable gene mutations in the test species.
(5) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, and paragraph (h) of this section, the following specific information shall be reported:
(i) Strain, age and weight of animals used, number of animals of each sex in experimental and control groups.
(ii) Test chemical vehicle, doses used and rationale for dose selection, toxicity data.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Time of examination for mutant progeny.
(vi) Criteria for scoring mutants.
(vii) Use of concurrent or negative controls.
(viii) Dose response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Russell, L.B., Shelby, P.B., von Halle, E., Sheridan, W., Valcovic, L. The mouse specific locus test with agents other than radiations: interpretation of data and recommendations for future work: A report of the U.S. EPA’s Gene-Tox Program,” Mutation Research, 86:329-354 (1981).
(2) [Reserved]
(h) Additional requirements. Testing facilities conducting the mouse visible specific locus test in accordance with this section shall, in addition to adhering to the provisions of §§ 792.190 and 792.195 of this chapter, obtain, and retain for at least 10 years, acceptable 35-mm color photographs (and their negatives) demonstrating the visible mutations observed in mutant animals and the lack of such mutations in their siblings and parents.
§ 798.5265 The salmonella typhimurium reverse mutation assay.
(a) Purpose. The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures his
(b) Definitions. (1) A reverse mutation assay in Salmonella typhimurium detects mutation in a gene of a histidine requiring strain to produce a histidine independent strain of this organism.
(2) Base pair mutagens are agents which cause a base change in the DNA. In a reversion assay, this change may occur at the site of the original mutation or at a second site in the chromosome.
(3) Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule.
(c) Reference substances. These may include, but need not be limited to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene, congo red, benzopurpurin 4B, trypan blue or direct blue 1.
(d) Test method – (1) Principle. Bacteria are exposed to test chemical with and without a metabolic activation system and plated onto minimal medium. After a suitable period of incubation, revertant colonies are counted and compared to the number of spontaneous revertants in an untreated and/or vehicle control culture.
(2) Description. Several methods for performing the test have been described. Among those used are:
(i) The direct plate incorporation method.
(ii) The preincubation method.
(iii) The azo-reduction method.
(3) Strain selection – (i) Designation. At the present time four strains, TA 1535, TA 1537, TA 98 and TA 100 should be used. The use of other strains in addition to these four is left to the discretion of the investigator.
(ii) Preparation and storage. Recognized methods of stock culture preparation and storage should be used. The requirement of histidine for growth should be demonstrated for each strain. Other phenotypic characteristics should be checked using such methods as crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency should be in the range expected either as reported in the literature or as established in the laboratory by historical control values.
(iii) Bacterial growth. Fresh cultures of bacteria should be grown up to the late exponential or early stationary phase of growth (approximately 10
(4) Metabolic activation. Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. For the direct plate incorporation method, the most commonly used system is a cofactor supplemented postmitochondrial fraction prepared from the livers of rodents treated with enzyme inducing agents such as Aroclor 1254. For the azo-reduction method, a cofactor supplemented postmitochondrial fraction prepared from the livers of untreated hamsters is preferred. For this method, the cofactor supplement should contain flavin mononucleotide, exogenous glucose 6-phosphate dehydrogenase, NADH and excess of glucose-6-phosphate.
(5) Control groups – (i) Concurrent controls. Concurrent positive and negative (untreated and/or vehicle) controls shall be included in each experiment. Positive controls shall ensure both strain responsiveness and efficacy of the metabolic activation system.
(ii) Strain specific positive controls. Strain specific positive controls shall be included in the assay. Examples of strain specific positive controls are as follows:
(A) Strain TA 1535, TA 100, sodium azide.
(B) TA 98, 2-nitrofluorene.
(C) TA 1537, 9-aminoacridine.
(iii) Positive controls to ensure the efficacy of the activation system. The positive control reference substance for tests including a metabolic activation system should be selected on the basis of the type of activation system used in the test. 2-Aminoanthracene is an example of a positive control compound in plate-incorporation tests using postmitochondrial fractions from the livers of rodents treated with enzyme inducing agents such as Aroclor-1254. Congo red is an example of a positive control compound in the azo-reduction method. Other positive control reference substances may be used.
(iv) Class-specific positive controls. The azo-reduction method should include positive controls from the same class of compounds as the test agent wherever possible.
(6) Test chemicals – (i) Vehicle. Test chemicals and positive control reference substances should be dissolved or suspended in an appropriate vehicle and then further diluted in vehicle for use in the assay.
(ii) Exposure concentrations. (A) The test should initially be performed over a broad range of concentrations. Among the criteria to be taken into consideration for determining the upper limits of test chemical concentration are cytotoxicity and solubility. Cytotoxicity of the test chemical may be altered in the presence of metabolic activation systems. Toxicity may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the background lawn or by the degree of survival of treated cultures. Relatively insoluble compounds should be tested up to the limits of solubility. For freely soluble nontoxic chemicals, the upper test chemical concentration should be determined on a case by case basis.
(B) Generally, a maximum of 5 mg/plate for pure substances is considered acceptable. At least 5 different amounts of test substance shall be tested with adequate intervals between test points.
(C) When appropriate, a single positive response shall be confirmed by testing over a narrow range of concentrations.
(e) Test performance – (1) Direct plate incorporation method. For this test without metabolic activation, test chemica1 and 0.1 m1 of a fresh bacterial culture should be added to 2.0 ml of overlay agar. For tests with metabolic activation, 0.5 ml of activation mixture containing an adequate amount of postmitochondrial fraction should be added to the agar overlay after the addition of test chemical and bacteria. Contents of each tube shall be mixed and poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted.
(2) Azo-reduction method. (i) For this test with metabolic activation, 0.5 ml of S-9 mix containing 150 ul of S-9 and 0.1 ml of bacterial culture should be added to a test tube kept on ice. One-tenth milliliter of chemical should be added, and the tubes should be incubated with shaking at 30 °C for 30 min. At the end of the incubation period, 2.0 ml of agar should be added to each tube, the contents mixed and poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted.
(ii) For tests without metabolic activation, 0.5 ml of buffer should be used in place of the 0.5 ml of S-9 mix. All other procedures shall be the same as those used for the test with metabolic activation.
(3) Other methods. Other methods may also be appropriate.
(4) Media. An appropriate selective medium with an adequate overlay agar shall be used.
(5) Incubation conditions. All plates within a given experiment shall be incubated for the same time period. This incubation period shall be for 48-72 hours at 37 °C.
(6) Number of cultures. All plating should be done at least in triplicate.
(f) Data and report – (1) Treatment of results. Data shall be presented as number of revertant colonies per plate for each replicate and dose. The numbers of revertant colonies on both negative (untreated and/or vehicle) and positive control plates shall also be presented. Individual plate counts, the mean number of revertant colonies per plate and standard deviation shall be presented for test chemical and positive and negative (untreated and/or vehicle) controls.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of revertants. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of revertants or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results from the S. typhimurium reverse mutation assay indicate that, under the test conditions, the test substance induces point mutations by base changes or frameshifts in the genome of this organism.
(ii) Negative results indicate that under the test conditions the test substance is not mutagenic in S. typhimurium.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Bacterial strain used.
(ii) Metabolic activation system used (source, amount and cofactor); details of preparations of S-9 mix.
(iii) Dose levels and rationale for selection of dose.
(iv) Positive and negative controls.
(v) Individual plate counts, mean number of revertant colonies per plate, standard deviation.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test,” Mutation Research 31:347-364 (1975).
(2) de Serres, F.J., Shelby, M.D. “The Salmonella mutagenicity assay: recommendations,” Science 203:563-565 (1979).
(3) Prival, M.J., Mitchell, V.D. “Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S-9,” Mutation Research 97:103-116 (1982).
(4) Vogel, H.J., Bonner, D.M. “Acetylornithinase of E. coli: partial purification and some properties,” Journal of Biological Chemistry. 218:97-106 (1956).
§ 798.5275 Sex-linked recessive lethal test in drosophila melanogaster.
(a) Purpose. The sex-linked recessive lethal (SLRL) test using Drosophila melanogaster detects the occurrence of mutations, both point mutations and small deletions, in the germ line of the insect. This test is a forward mutation assay capable of screening for mutations at about 800 loci on the X-chromosome. This represents about 80 percent of all X-chromosome loci. The X-chromosome represents approximately one-fifth of the entire haploid genome.
(b) Definitions. (1) Lethal mutation is a change in the genome which, when expressed, causes death to the carrier.
(2) Recessive mutation is a change in the genome which is expressed in the homozygous or hemizygous condition.
(3) Sex-Linked genes are present on the sex (X or Y) chromosomes. Sex-linked genes in the context of this guideline refer only to those located on the X-chromosome.
(c) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate or N-nitroso-dimethylamine.
(d) Test method – (1) Principle. Mutations in the X-chromosome of D. melanogaster are phenotypically expressed in males carrying the mutant gene. When the mutation is lethal in the hemizygous condition, its presence is inferred from the absence of one class of male offspring out of the two that are normally produced by a heterozygous female. The SLRL test takes advantage of these facts by means of specially marked and arranged chromosomes.
(2) Description. Wild-type males are treated and mated to appropriate females. Female offspring are mated individually to their brothers, and in the next generation the progeny from each separate dose are scored for phenotypically wild-type males. Absence of these males indicates that a sex-linked recessive lethal mutation has occurred in a germ cell of the P
(3) Drosophila stocks. Males of a well-defined wild type stock and females of the Muller-5 stock may be used. Other appropriately marked female stocks with multiple inverted X-chromosomes may also be used.
(4) Control groups – (i) Concurrent controls. Concurrent positive and negative (vehicle) controls shall be included in each experiment.
(ii) Positive controls. Examples of positive controls include ethyl methanesulfonate and N-nitroso-dimethylamine.
(iii) Other positive controls. Other positive control reference substances may be used.
(iv) Negative controls. Negative (vehicle) controls shall be included. The size of the negative (vehicle) control group shall be determined by the availability of appropriate laboratory historical control data.
(5) Test chemicals – (i) Vehicle. Test chemicals should be dissolved in water. Compounds which are insoluble in water may be dissolved or suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-60 or 80) and then diluted in water or saline prior to administration. Dimethylsulfoxide should br avoided as a vehicle.
(ii) Dose levels. For the initial assessment of mutagenicity, it is sufficient to test a single dose of the test substance for screening purposes. This dose should be the maximum tolerated dose, or that which produces some indication of toxicity, or shall be the highest dose attainable. For dose-response purposes, at least three additional dose levels should be used.
(iii) Route of administration. Exposure may be oral, by injection or by exposure to gases or vapors. Feeding of the test compound may be done in sugar solution. When necessary, substances may be dissolved in 0.7 percent NaCl solution and injected into the thorax or abdomen.
(e) Test performance – (1) Treatment and mating. Wild-type males (3 to 5 days old) shall be treated with the test substance and mated individually to an appropriate number of virgin females from the Muller-5 stock or females from another appropriately marked (with multiply-inverted X-chromosomes) stock. The females shall be replaced with fresh virgins every 2 to 3 days to cover the entire germ cell cycle. The offspring of these females are scored for lethal effects corresponding to the effects on mature sperm, mid or late stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment.
(2) F
(3) Number of matings. (i) The test should be designed with a predetermined sensitivity and power. The number of flies in each group should reflect these defined parameters. The spontaneous mutant frequency observed in the appropriate control group will strongly influence the number of treated chromosomes that must be analysed to detect substances which show mutation rates close to those of the controls.
(ii) Test results should be confirmed in a separate experiment.
(f) Data and report – (1) Treatment of results. Data shall be tabulated to show the number of chromosomes tested, the number of nonfertile males and the number of lethal chromosomes at each exposure concentration and for each mating period for each male treated. Numbers of clusters of different size per male shall be reported.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical techniques.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of sex-lined recessive lethals. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of sex-linked recessive lethals or a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the SLRL test in D. melanogaster indicate that under the test conditions the test agent causes mutations in germ cells of this insect.
(ii) Negative results indicate that under the test conditions the test substance is not mutagenic in D. melanogaster.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported.
(i) Drosophila stock used in the assay, age of insects, number of males treated, number of sterile males, number of F
(ii) Test chemical vehicle, treatment and sampling schedule, exposure levels, toxicity data, negative (vehicle) and positive controls, if appropriate.
(iii) Criteria for scoring lethals.
(iv) Number of chromosomes tested, number of chromosomes scored, number of chromosomes carrying a lethal mutation.
(v) Historical control data, if available.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Sobels, F.H., Vogel, E. “The capacity of Drosophila for detecting relevant genetic damage,” Mutation Research 41:95-106 (1976).
(2) Wurgler F.E., Sobels F.H., Vogel E. “Drosophila as assay system for detecting genetic changes,” Handbook of mutagenicity test procedures. Eds. Kilbey, B.J., Legator, M., Nichols, W., Ramel, C., (Amsterdam: Elsevier/North Holland Biomedical Press, 1977) pp. 335-373.
§ 798.5300 Detection of gene mutations in somatic cells in culture.
(a) Purpose. Mammalian cell culture systems may be used to detect mutations induced by chemical substances. Widely used cell lines include L5178Y mouse lymphoma cells and the CHO and V-79 lines of Chinese hamster cells. In these cell lines the most commonly used systems measure mutation at the thymidine kinase (TK), hypoxanthine-guanine-phosphoribosyl transferase (HPRT) and Na
(b) Definitions. (1) A forward mutation assay detects a gene mutation from the parental type to the mutant form which gives rise to a change in an enzymatic or functional protein.
(2) Base pair mutagens are agents which cause a base change in the DNA.
(3) Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule.
(4) Phenotypic expression time is a period during which unaltered gene products are depleted from newly mutated cells.
(c) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate, N-nitroso-dimethylamine, 2-acetylaminofluorene, 7,12-dimethylbenzanthracene or hycanthone.
(d) Test method – (1) Principle. Cells are exposed to test substance, both with and without metabolic activation, for a suitable period of time and subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cells deficient in thymidine kinase (TK) due to the forward mutation TK
(2) Description. Cells in suspension or monolayer culture are exposed to the test substance, both with and without metabolic activation, for a defined period of time. Cytotoxicity is determined by measuring the colony forming ability or growth rate of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time – characteristic of each selected locus – to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency. After a suitable incubation time, cell colonies are counted. The number of mutant colonies in selective medium is adjusted by the number of colonies in nonselective medium to derive the mutant frequency.
(3) Cells – (i) Type of cells used in the assay. A variety of cell lines are available for use in this assay including subclones of L5178Y, CHO cells or V-79 cells. Cell types used in this assay should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a low spontaneous mutation frequency. Cells should be checked for Mycoplasma contamination and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media and incubation conditions (culture vessels, CO
(4) Metabolic activation. Cells shall be exposed to test substance both in the presence and absence of an appropriate metabolic activation system.
(5) Control groups. Positive and negative (untreated and/or vehicle) controls shall be included in each experiment. When metabolic activation is used, the positive control substance shall be known to require such activation.
(6) Test chemicals – (i) Vehicle. Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of the vehicle shall not interfere with cell viability or growth rate. Treatment vessels should be chosen to ensure that there is no visible interaction, such as etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. (A) The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures, and concentrations of test substance used should reflect these defined parameters. The number of cells per culture is based on the expected background mutant frequency; a general guide is to use a number which is 10 times the inverse of this frequency.
(B) Several concentrations (usually at least 4) of the test substance shall be used. Generally, these shall yield a concentration-related toxic effect. The highest concentration shall produce a low level of survival (approximately 10 percent), and the survival in the lowest concentration shall approximate the negative control. Cytotoxicity shall be determined after treatment with the test substance both in the presence and in the absence of an exogenous metabolic activation system. Relatively insoluble substances should be tested up to their limit of solubility under culture conditions. For freely-soluble nontoxic substances the highest concentration used should be determined on a case-by-case basis.
(e) Test performance. (1) Cells shall be exposed to the test substance both with and without exogenous metabolic activation. Exposure shall be for a suitable period of time, in most cases 1 to 5 hours is effective; exposure time may be extended over one or more cell cycles.
(2) At the end of the exposure period, cells shall be washed and cultured to determine viability and to allow for expression of the mutant phenotype.
(3) At the end of the expression period, which shall be sufficient to allow near optimal phenotypic expression of induced mutants, cells should be grown in medium with and without selective agent(s) for determination of number of mutants and cloning efficiency, respectively.
(4) Results shall be confirmed in an independent experiment. When appropriate, a single positive response should be confirmed by testing over a narrow range of concentrations.
(f) Data and report – (1) Treatment of results. Data shall be presented in tabular form. Individual colony counts for the treated and control groups shall be presented for both mutation induction and survival. Survival and cloning efficiencies shall be given as a percentage of the controls. Mutant frequency shall be expressed as number of mutants per number of surviving cells.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant concentration-related increase in the mutant frequency. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant concentration-related increase in the mutant frequency or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results for an in vitro mammalian cell gene mutation test indicate that, under the test conditions, a substance induces gene mutations in the cultured mammalian cells used.
(ii) Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Cell type used, number of cell cultures, methods used for maintenance of cell cultures.
(ii) Rationale for selection of concentrations and number of cultures.
(iii) Test conditions: composition of media, CO
(iv) Methods used to enumerate numbers of viable and mutant cells.
(v) Dose-response relationship, where possible.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Amacher, D.E., Paillet, S.C., Ray, V. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicology testing,” Mutation Research, 64:391-406 (1979).
(2) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. Salsburg, V.A. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation,” Mutation Research, 72:447-474 (1980).
(3) Bradley, M.O., Bhuyan B., Francis, M.C., Langenback, R., Peterson, A., Huberman, E. “Mutagenesis by chemical agents in V-79 Chinese hamster cells: a review and analysis of the literature: a report of the Gene-Tox Program,” Mutation Research, 87:81-142 (1981).
(4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown, M.M. “Validation and characterization of the L5178Y TK
(5) Clive, D., Spector, J.F.S. “Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells,” Mutation Research, 31:17-29 (1975).
(6) Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O’Neill, J.P., Whitfield, B.L. “The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals: a report of the U.S. EPA’s Gene-Tox Program,” Mutation Research, 86:193-214 (1981).
§ 798.5375 In vitro mammalian cytogenetics.
(a) Purpose. The in vitro cytogenetics test is a mutagenicity test system for the detection of chromosomal aberrations in cultured mammalian cells. Chromosomal aberrations may be either structural or numerical. However, because cytogenetic assays are usually designed to analyse cells at their first post-treatment mitosis and numerical aberrations require at least one cell division to be visualized, this type of aberration is generally not observed in a routine cytogenetics assay. Structural aberrations may be of two types, chromosome or chromatid.
(b) Definitions. (1) Chromosome-type aberrations are changes which result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method – (1) Principle. In vitro cytogenetics assays may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance both with and without metabolic activation. Following exposure of cell cultures to test substances, they are treated with a spindle inhibitor (e.g., colchicine or Colcemid#) to arrest cells in a metaphase-like stage of mitosis (c-metaphase). Cells are then harvested and chromosome preparations made. Preparations are stained and metaphase cells are analyzed for chromosomal aberrations.
(2) Description. Cell cultures are exposed to test compounds and harvested at various intervals after treatment. Prior to harvesting, cells are treated with a spindle inhibitor (e.g., colchicine or Colcemid#) to accumulate cells in c-metaphase. Chromosome preparations from cells are made, stained and scored for chromosomal aberrations.
(3) Cells – (i) Type of cells used in the assay. There are a variety of cell lines or primary cell cultures, including human cells, which may be used in the assay. Established cell lines and strains should be checked for Mycoplasma contamination and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media, and incubation conditions (culture vessels CO
(4) Metabolic activation. Cells shall be exposed to test substance both in the presence and absence of an appropriate metabolic activation system.
(5) Control groups. Positive and negative (untreated and/or vehicle) controls both with and without metabolic activation shall be included in each experiment. When metabolic activation is used, the positive control substance shall be known to require such activation.
(6) Test chemicals – (i) Vehicle. Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. Final concentration of the vehicle shall not interfere with cell viability or growth rate. Treatment vessels should be chosen to ensure that there is no visible interaction, such as etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. Multiple concentrations of the test substance over a range adequate to define the response should be tested. Generally the highest test substance concentrations tested with and without metabolic activation should show evidence of cytotoxicity or reduced mitotic activity. Relatively insoluble substances should be tested up to the limit of solubility. For freely soluble nontoxic chemicals, the upper test chemical concentration should be determined on a case by case basis.
(e) Test performance – (1) Established cell lines and strains. Prior to use in the assay, cells should be generated from stock cultures, seeded in culture vessels at the appropriate density and incubated at 37 °C.
(2) Human lymphocyte cultures. Heparinized or acid-citrate-dextrose whole blood should be added to culture medium containing a mitogen, e.g., phytohemagglutinin (PHA) and incubated at 37 °C. White cells sedimented by gravity (buffy coat) or lymphocytes which have been purified on a density gradient may also be utilized.
(3) Treatment with test substance. For established cell lines and strains, cells in the exponential phase of growth shall be treated with test substances in the presence and absence of an exogenous metabolic activation system. Mitogen-stimulated human lymphocyte cultures may be treated with the test substance in a similar manner.
(4) Number of cultures. At least two independent cultures shall be used for each experimental point.
(5) Culture harvest time. (i) For established cell lines and strains, multiple harvest times are recommended. However, for screening purposes, a single harvest time may be appropriate. If the test chemical changes the cell cycle length, the fixation intervals should be changed accordingly. If a single harvest time is selected, supporting data for the harvest time should be presented in such a study.
(ii) For human lymphocyte cultures, the substance to be tested may be added to the cultures at various times after mitogen stimulation so that there is a single harvest time after the initiation of the cell culture. Alternatively, a single treatment may be followed by multiple harvest times. Harvest time should be extended for those chemicals which induce an apparent cell cycle delay. Because the population of human lymphocytes is only partially synchronized, a single treatment, at, or close to, the time when metaphase stages first appear in the culture will include cells in all phases of the division cycle. Therefore, a single harvest at the time of second mitosis may be carried out for screening purposes.
(iii) Cell cultures shall be treated with a spindle inhibitor, (e.g., colchicine or Colcemid ®), 1 or 2 hours prior to harvesting. Each culture shall be harvested and processed separately for the preparation of chromosomes.
(6) Chromosome preparation. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining.
(7) Analysis. Slides shall be coded before analysis. In human lymphocytes, only cells containing 46 centromeres shall be analyzed. In established cell lines and strains, only metaphases containing ±2 centromeres of the modal number shall be analyzed. Uniform criteria for scoring aberrations shall be used.
(8) Confirmatory tests. When appropriate, a single positive response shall be confirmed by testing over a narrow range of concentrations.
(f) Data and report – (1) Treatment of results. Data shall be presented in a tabular form. Different types of structural chromosomal aberrations shall be listed with their numbers and frequencies for experimental and control groups. Data should be evaluated by appropriate statistical methods. Gaps or achromatic lesions are recorded separately and not included in the total aberration frequency.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of structural chromosomal aberrations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of structural chromosomal aberrations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the in vitro cytogenetics assay indicate that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
(ii) Negative results indicate that under the test conditions the test substance does not induce chromosomal aberrations in cultured mammalian somatic cells.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Cells used, density and passage number at time of treatment, number of cell cultures.
(ii) Methods used for maintenance of cell cultures including medium, temperature and CO
(iii) Test chemical vehicle, concentration and rationale for the selection of the concentrations used in the assay, duration of treatment.
(iv) Details of both the protocol used to prepare the metabolic activation system and of its use in the assay.
(v) Identity of spindle inhibitor, its concentration and duration of treatment.
(vi) Date of cell harvest.
(vii) Positive and negative controls.
(viii) Methods used for preparation of slides for microscopic examination.
(ix) Number of metaphases analysed.
(x) Mitotic index where applicable.
(xi) Criteria for scoring aberrations.
(xii) Type and number of aberrations, given separately for each treated and control culture, total number of aberrations per group; frequency distribution of number of chromosomes in established cell lines and strains.
(xiii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted.
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test,” Mutation Research, 31:347-364 (1975).
(2) Evans, H.J. “Cytological methods for detecting chemical mutagens,” Chemical mutagens, principles and methods for their detection, Vol. 4, Ed. A. Hollaender (New York, London: Plenum Press, 1976) pp. 1-29.
(3) Howard, P.N., Bloom, A.D., Krooth, R.S. “Chromosomal aberrations induced by N-methyl-N′-nitro-N-nitrosoguanidine in mammalian cells,” In Vitro 7:359-365 (1972).
(4) Ishidate, M. Jr., Odashima, S. “Chromosome tests with 134 compounds on Chinese hamster cells in vitro: A screening for chemical carcinogens,” Mutation Research, 48:337-354 (1975).
(5) Preston, R.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J.S., “Mammalian in vivo and in vitro cytogenetic assays: A report of the Gene-tox Program,” Mutation Research, 87:143-188 (1981).
§ 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.
(a) Purpose. The in vivo bone marrow cytogenetic test is a mutagenicity test for the detection of structural chromosomal aberrations. Chromosomal aberrations are generally evaluated in first post-treatment mitoses. With the majority of chemical mutagens, induced aberrations are of the chromatid type but chromosome type aberrations also occur.
(b) Definitions. (1) Chromosome-type aberrations are changes which result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method – (1) Principle. Animals are exposed to test chemicals by appropriate routes and are sacrificed at sequential intervals. Chromosome preparations are made from bone marrow cells. The stained preparations are examined and metaphase cells are scored for chromosomal aberrations.
(2) Description. The method employs bone marrow of laboratory rodents which have been exposed to test chemicals. Prior to sacrifice, animals are further treated with a spindle inhibitor, (e.g., colchicine or Colcemid ®) to arrest the cells in c-metaphase. Chromosome preparations from the cells are stained and scored for chromosomal aberrations.
(3) Animal selection – (i) Species and strain. Any appropriate mammalian species may be used. Examples of commonly used rodent species are rats, mice, and hamsters.
(ii) Age. Healthy young adult animals shall be used.
(iii) Number and sex. At least five female and five male animals per experimental and control group shall be used. Thus, 10 animals would be sacrificed per time per group treated with the test compound if several test times after treatment are included in the experimental schedule. The use of a single sex or smaller number of animals should be justified.
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups – (1) Concurrent controls. (i) Concurrent positive and negative (vehicle) controls shall be included in the assay.
(ii) Positive controls. A single dose positive control showing a significant response at any one time point is adequate. A compound known to produce chromosomal aberrations in vivo shall be employed as the positive control.
(5) Test chemicals – (i) Vehicle. When possible, test chemicals shall be dissolved in isotonic saline or distilled water. Water insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicles used shall neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test compound should be employed.
(ii) Dose levels. For an initial assessment, one dose of the test substance may be used, the dose being the maximum tolerated dose (to a maximum of 5,000 mg/kg) or that producing some indication of cytotoxicity (e.g., partial inhibition of mitosis) or shall be the highest dose attainable (to a maximum of 5,000 mg/kg). Additional dose levels may be used. For determination of dose-response, at least three dose levels should be used.
(iii) Route of administration. The usual routes are oral or by intraperitoneal injection. Other routes may be appropriate.
(iv) Treatment schedule. In general, test substances should be administered once only. However, based on toxicological information a repeated treatment schedule may be employed.
(e) Test performance – (1) Generally the test may be performed in two assays. (i) Animals should be treated with the test substance once at the selected dose(s). Samples should be taken at three times after treatment. For rodents, the central sampling interval is 24 hours. Since cell cycle kinetics can be influenced by the test substance, one earlier and one later sampling interval adequately spaced within the range of 6 to 48 hours shall be applied. Where the additional dose levels are tested in a subsequent experiment, samples shall be taken at the predetermined most sensitive interval or, if this is not established, at the central sampling time. If the most sensitive interval is known and documented with data, only this one time point shall be sampled.
(ii) If a repeated treatment schedule is used at the selected dose(s), samples shall be taken 6 and 24 hours after the last treatment; other sampling times may be used if justified. Where the additional dose levels are tested in a subsequent experiment, samples shall be taken at the predetermined most sensitive interval or, if this is not established, at 6 hours after the last treatment.
(2) Administration of spindle inhibitor. Prior to sacrifice, animals shall be injected IP with an appropriate dose of a spindle inhibitor (e.g., colchicine or Colcemid ®) to arrest cells in c-metaphase.
(3) Preparation of slides. Immediately after sacrifice, the bone marrow shall be obtained, exposed to hypotonic solution, and fixed. The cells shall then be spread on slides and stained. Chromosome preparations shall be made following standard procedures.
(4) Analysis. The number of cells to be analyzed per animal should be based upon the number of animals used, the negative control frequency, the predetermined sensitivity, and the power chosen for the test. Slides shall be coded before microscopic analysis.
(f) Data and report – (1) Treatment of results. Data should be presented in tabular form for both cells and animals. Different types of structural chromosomal aberrations should be listed with their numbers and a mean frequency per cell for each animal in all treated and control groups. Gaps (achromatic lesions) should be recorded separately and not included in the total abberration frequency. Differences among animals within each group should be considered before making comparisons between treated and control groups.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of structual chromosomal aberrations or abnormal metaphase figures. Another criterion may be based upon detection of a reproducible and statistically significant positive response for a least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of chromosomal aberrations or abnormal metaphase figures or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the in vivo bone marrow cytogenetics assay indicate that under the test conditions the test substance induces chromosomal aberrations in the bone marrow of the test species.
(ii) Negative results indicate that under the test conditions, the test substance does not induce chromosomal aberrations in the bone marrow of the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Species, strain, age, weight, number and sex of animals in each treatment and control group.
(ii) Test chemical vehicle, dose levels used, rationale for dose selection.
(iii) Route of administration, treatment and sampling schedules, toxicity data, negative and positive controls.
(iv) Identity of spindle-inhibitor, its concentration and duration of treatment.
(v) Details of the protocol used for chromosome preparation, number of cells scored per animal, type and number of aberrations given separately for each treated and control animal.
(vi) Mitotic index, where applicable.
(vii) Criteria for scoring aberrations.
(viii) Number and frequency of aberrant cells per animal in each treatment and control groups.
(ix) Total number of aberrations per group.
(x) Number of cells with aberrations per group.
(xi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Adler, I.D., Ramarao, G., Epstein, S.S. “In vivo cytogenetic effects of trimethyl-phosphate and of TEPA on bone marrow cells of male rats,” Mutation Research, 13:263-273 (1971).
(2) Evans, H.J. “Cytological methods for detecting chemical mutagens,” Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4. Ed. A. Hollaender (New York and London: Plenum Press, 1976) pp. 1-29.
(3) Kilian, J.D., Moreland, F.E. Benge, M.C., Legator, M.S., Whorton, E.B. Jr. “A collaborative study to measure intralaboratory variation with the in vivo bone morrow metaphase procedure,” Handbook of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M. Nichols, C., Ramel, D., (Amsterdam: Elsevier/North Holland Biomedical Press, 1977) 243-260.
(4) Preston, J.R., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V. Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981).
§ 798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.
(a) Purpose. The micronucleus test is a mammalian in vivo test which detects damage of the chromosomes or mitotic apparatus by chemicals. Polychromatic erythrocytes in the bone marrow of rodents are used in this assay. When the erythroblast develops into an erythrocyte the main nucleus is extruded and may leave a micronucleus in the cytoplasm. The visualization of micronuclei is facilitated in these cells because they lack a nucleus. Micronuclei form under normal conditions. The assay is based on an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow of treated animals.
(b) Definition. Micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes, which lag behind at anaphase of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.
(c) Reference substances. Not applicable.
(d) Test method – (1) Principle. (i) Animals are exposed to test substance by an appropriate route. They are sacrificed, the bone marrow extracted and smear preparations made and stained. Polychromatic erythrocytes are scored for micronuclei under the microscope.
(ii) Micronuclei may also be detected in other test systems:
(A) Tissue culture.
(B) Plants.
(C) Blood smears.
(D) Fetal tissues.
(E) Meiotic cells.
(F) Hepatic cells.
(iii) The present guideline is based on the mammalian bone marrow assay.
(2) Description. The method employs bone marrow of laboratory mammals which are exposed to test substances.
(3) Animal selection – (i) Species and strain. Mice are recommended. However, any appropriate mammalian species may be used.
(ii) Age. Young adult animals shall be used.
(iii) Number and sex. At least five female and five male animals per experimental and control group shall be used. Thus, 10 animals would be sacrificed per time per group if several test times after treatment were included in the experimental schedule. The use of a single sex or a smaller number of animals should be justified.
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups – (i) Concurrent controls. Concurrent positive and negative (vehicle) controls shall be included in each assay.
(ii) Positive controls. A compound known to produce micronuclei in vivo shall be employed as the positive control.
(5) Test chemicals – (i) Vehicle. When appropriate for the route of administration, solid and liquid test substances should be dissolved or suspended in distilled water or isotonic saline. Water insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test compound nor produce toxic effects. Fresh preparations of the test compound should be employed.
(ii) Dose levels. For an initial assessment, one dose of the test substance may be used, the dose being the maximum tolerated dose (to a maximum of 5,000 mg/kg) or that producing some indication of cytotoxicity, e.g., a change in the ratio of polychromatic to normochromatic erythrocytes. Additional dose levels may be used. For determination of dose response, at least three dose levels shall be used.
(iii) Route of administration. The usual routes of administration are IP or oral. Other routes may be appropriate.
(iv) Treatment schedule. Test substances should generally be administered only once. However, based upon toxicological information a repeated treatment schedule may be employed.
(e) Test performance – (1) Treatment and sampling times. (i) Animals shall be treated with the test substance once at the highest tolerated dose. Sampling times should coincide with the maximum responses of the assay which varies with the test substance. Therefore, using the highest dose, bone marrow samples should be taken at least three times, starting not earlier than 12 hours after treatment, with appropriate intervals following the first sample but not extending beyond 72 hours. When other doses are used sampling shall be at the maximum sensitive period, or, if that is not known, approximately 24 hours after treatment. Other appropriate sampling times may be used in addition. If the most sensitive interval is known and documented with data, only this one time point need be sampled.
(ii) If a repeated treatment schedule is used, samples shall be taken at least three times, starting not earlier than 12 hours after the last treatment and at appropriate intervals following the first sample, but not extending beyond 72 hours.
(iii) Bone marrow shall be obtained immediately after sacrifice. Cells shall be prepared, put on slides, spread as a smear and stained.
(2) Analysis. Slides shall be coded before microscopic analysis. At least 1,000 polychromatic erythrocytes per animal shall be scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes should be determined for each animal by counting a total of 200 erythrocytes. To ensure consistency with OECD and other guidelines, 1,000 polychromatic erythrocytes are recommended. Additional information may be obtained by scoring normochromatic erythrocytes for micronuclei.
(f) Data and report – (1) Treatment of results. Criteria for scoring micronuclei shall be given. Individual data shall be presented in a tabular form including positive and negative (vehicle) controls and experimental groups. The number of polychromatic erythrocytes scored, the number of micronucleated polychromatic erythrocytes, the percentage of micronucleated cells, the number of micronucleated normochromatic erythrocytes, and, if applicable, the percentage of micronucleated erythrocytes and the ratio of normochromatic to polychromatic erythrocytes shall be listed separately for each experimental and control animal. Absolute numbers shall be included if percentages are reported.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive response, one of which is a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) The results of the micronucleus test provide information on the ability of a chemical to induce micronuclei in polychromatic erythrocytes of the test species under the conditions of the test. This damage may have been the result of chromosomal damage or damage to the mitotic apparatus.
(ii) Negative results indicate that under the test conditions the test substance does not produce micronuclei in the bone marrow of the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Species, strain, age, weight, number and sex of animals in each treatment and control group.
(ii) Test chemical vehicle, dose levels used, rationale for dose selection.
(iii) Rationale for and description of treatment and sampling schedules, toxicity data, negative and positive controls.
(iv) Details of the protocol used for slide preparation.
(v) Criteria for identifying micronucleated erythrocytes.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Cihak, R. “Evaluation of benzidine by the micronucleus test,” Mutation Research, 67: 383-384 (1979).
(2) Cole, R.J., Taylor, N., Cole, J., Arlett, C.F. “Short-term tests for transplacentally active carcinogens. 1. Micronucleus formation in fetal and maternal mouse erythroblasts,” Mutation Research, 80: 141-157 (1981).
(3) Kliesch, U., Danford, N., Adler, I.D. “Micronucleus test and bone-marrow chromosome analysis. A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations,” Mutation Research, 80: 321-332 (1981).
(4) Matter, B., Schmid, W. “Trenimon-induced chromosomal damage in bone-marrow cells of six mammalian species, evaluated by the micronucleus test,” Mutation Research, 12: 417-425 (1971).
(5) Schmid, W. “The micronucleus test,” Mutation Research, 31:9-15 (1975).
(6) Schmid, W. “The micronucleus test for cytogenetic analysis,” Chemical Mutagens, Principles and Methods for their Detection. Vol. 4 Hollaender A, (Ed. A ed. (New York and London: Plenum Press, (1976) pp. 31-53.
(7) Heddle, J.A., Hite, M., Kurkhart, B., Mavournin, K., MacGregor, J.T., Newell, G.W., Salamone, M.F. “The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program,” Mutation Research, 123: 61-118 (1983).
§ 798.5450 Rodent dominant lethal assay.
(a) Purpose. Dominant lethal (DL) effects cause embryonic or fetal death. Induction of a dominant lethal event after exposure to a chemical substance indicates that the substance has affected germinal tissue of the test species. Dominant lethals are generally accepted to be the result of chromosomal damage (structural and numerical anomalies) but gene mutations and toxic effects cannot be excluded.
(b) Definition. A dominant lethal mutation is one occurring in a germ cell which does not cause dysfunction of the gamete, but which is lethal to the fertilized egg or developing embryo.
(c) Reference substances. These may include, but need not be limited to, triethylenemelamine, cyclophosphamide or ethyl methanesulfonate.
(d) Test method – (1) Principle. Generally, male animals are exposed to the test substance and mated to untreated virgin females. The various germ cell stages can be tested separately by the use of sequential mating intervals. The females are sacrificed after an appropriate period of time and the contents of the uteri are examined to determine the numbers of implants and live and dead embryos. The calculation of the dominant lethal effect is based on comparison of the live implants per female in the treated group to the live implants per female in the control group. The increase of dead implants per female in the treated group over the dead implants per female in the control group reflects the post-implantation loss. The post-implantation loss is calculated by determining the ratio of dead to total implants from the treated group compared to the ratio of dead to total implants from the control group. Pre-implantation loss can be estimated on the basis of corpora lutea counts or by comparing the total implants per female in treated and control groups.
(2) Description. (i) Several treatment schedules are available. The most widely used requires single administration of the test substance. Other treatment schedules, such as treatment on five consecutive days, may be used if justified by the investigator.
(ii) Individual males are mated sequentially to virgin females at appropriate intervals. The number of matings following treatment is governed by the treatment schedule and should ensure that germ cell maturation is adequately covered. Females are sacrificed in the second half of pregnancy and the uterine contents examined to determine the total number of implants and the number of live and dead embryos.
(3) Animal selection – (i) Species. Rats or mice are generally used as the test species. Strains with low background dominant lethality, high pregnancy frequency and high implant numbers are recommended.
(ii) Age. Healthy, sexually mature animals shall be used.
(iii) Number. An adequate number of animals shall be used taking into account the spontaneous variation of the biological characteristics being evaluated. The number chosen should be based on the predetermined sensitivity of detection and power of significance. For example, in a typical experiment, the number of males in each group shall be sufficient to provide between 30 and 50 pregnant females per mating interval.
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups – (i) Concurrent controls. Generally concurrent positive and negative (vehicle) controls shall be included in each experiment. When acceptable positive control results are available from experiments conducted recently (within the last 12 months) in the same laboratory these results can be used instead of a concurrent positive control.
(ii) Positive controls. Positive control substances shall be used at a dose which demonstrates the test sensitivity.
(5) Test chemicals – (i) Vehicle. When possible, test substances shall be dissolved or suspended in isotonic saline or distilled water. Water-insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. Normally, three dose levels shall be used. The highest dose shall produce signs of toxicity (e.g., slightly reduced fertility and slightly reduced body weight). However, in an initial assessment of dominant lethality a single high dose may be sufficient. Nontoxic substances shall be tested at 5g/kg or, if this is not practicable, then as the highest dose attainable.
(iii) Route of administration. The usual routes of administration are oral or by IP injection. Other routes may be appropriate.
(e) Test performance. (1) Individual males are mated sequentially at appropriate predetermined intervals to one or two virgin females. Females should be left with the males for at least the duration of one estrus cycle or alternatively until mating has occurred as determined by the presence of sperm in the vagina or by the presence of a vaginal plug.
(2) The number of matings following treatment should be governed by the treatment schedule and should ensure that germ cell maturation is adequately covered.
(3) Females should be sacrificed in the second half of pregnancy and uterine contents examined to determine the number of implants and live and dead embryos. The ovaries may be examined to determine the number of corpora lutea.
(f) Data and report – (1) Treatment of results. Data shall be tabulated to show the number of males, the number of pregnant females, and the number of nonpregnant females. Results of each mating, including the identity of each male and female, shall be reported individually. For each female, the dose level and week of mating and the frequencies of live implants and of dead implants shall be enumerated. If the data are recorded as early and late deaths, the tables shall make that clear. If preimplantation loss is estimated, it shall be reported. Preimplantation loss can be calculated as the difference between the number of corpora lutea and the number of implants or as a reduction in the average number of implants per female in comparison with control matings.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods. Differences among animals within the control and treatment groups shall be considered before making comparisons between treated and control groups.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of dominant lethals. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of dominant lethals or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) A positive DL assay suggests that under the test conditions the test substance may be genotoxic in the germ cells of the treated sex of the test species.
(ii) A negative result suggests that under the conditions of the test the test substance may not be genotoxic in the germ cells of the treated sex of the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Species, strain, age and weights of animals used, number of animals of each sex in experimental and control groups.
(ii) Test substance, vehicle used, dose levels and rationale for dosage selection, negative (vehicle) and positive controls, experimental observations, including signs of toxicity.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Methods used to determine that mating has occurred (where applicable).
(vi) Criteria for scoring dominant lethals including the number of early and late embryonic deaths.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Brewen, J.G., Payne, H.S., Jones, K.P., Preston, R.J. “Studies on chemically induced dominant lethality. I. The cytogenetic basis of MMS-induced dominant lethality in post-meiotic germ cells” Mutation Research, 33:239-250 (1975).
(2) Ehling, U.H., Machemer, L., Buselmaier, E., Dycka, D., Frohberg, H., Kratochvilova, J., Lang, R., Lorke, D., Muller, D., Pheh, J., Rohrborn, G., Roll, R., Schulze-Schencking, M., Wiemann, H. “Standard protocol for the dominant lethal test on male mice. Set up by the Work Group “Dominant lethal mutations of the ad hoc Committee Chemogenetics,” Archives of Toxicology, 39:173-185 (1978).
§ 798.5460 Rodent heritable translocation assays.
(a) Purpose. This test detects transmitted chromosomal damage which manifests as balanced reciprocal translocations in progeny descended from parental males treated with chemical mutagens.
(b) Definitions. (1) A heritable translocation is one in which distal segments of nonhomologous chromosomes are involved in a reciprocal exchange.
(2) Diakinesis and metaphase I are stages of meiotic prophase scored cytologically for the presence of multivalent chromosome association characteristic of translocation carriers.
(c) Reference substances. Not applicable.
(d) Test method – (1) Principle. When a balanced reciprocal translocation is induced in a parental male germ cell, the resulting progeny is translocation heterozygote.
(i) Basis for fertility screening. Male translocation heterozygotes may be completely sterile. This class consists of two types of translocations:
(A) Translocations between non-homologous chromosomes in which at least one of the breaks occurs close to one end of a chromosome.
(B) Those that carry multiple translocations. The majority of male translocation heterozygotes are semisterile – they carry one or (rarely) two translocations. The degree of semisterility is dependent upon the proportions of balanced and unbalanced (duplication-deficiency) gametes produced in the ejaculate as a function of meiotic segregation. Balanced and unbalanced sperm are equally capable of fertilizing an egg. Balanced sperm lead to viable progeny. Unbalanced sperm result in early embryonic lethality.
(ii) Basis for cytological screening. The great majority of male translocation heterozygotes can be identified cytologically through analysis of diakinesis metaphase I spermatocytes. Translocation heterozygotes are characterized by the presence of multivalent chromosome association such as a ring or chain of four chromosomes held together by chiasmata in paired homologous regions. Some translocation carriers can be identified by the presence of extra long and/or extra short chromosomes in spermatogonial and somatic cell metaphase preparations.
(2) Description. Essentially, two methods have been used to screen for translocation heterozygosity; one method uses a mating sequence to identify sterile and semisterile males followed by cytological examination of suspect male individuals; the other method deletes the mating sequence altogether and all F
(3) Animal selection – (i) Species. The mouse is the species generally used, and is recommended.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. (A) The number of male animals necessary is determined by the following factors:
(1) The use of either historical or concurrent controls.
(2) The power of the test.
(3) The minimal rate of induction required.
(4) Whether positive controls are used.
(5) The level of significance desired.
(B) [Reserved]
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups – (i) Concurrent controls. No concurrent positive or negative (vehicle) controls are recommended as routine parts of the heritable translocation assay. However, investigators not experienced in performing translocation testing shall include a substance known to produce translocations in the assay as a positive control reference chemical.
(ii) Historical controls. At the present time, historical control data must be used in tests for significance. When statistically reliable historical controls are not available, negative (vehicle) controls shall be used.
(5) Test chemicals – (i) Vehicle. When appropriate for the route of administration, solid and liquid test substances should be dissolved or suspended in distilled water or isotonic saline. Water-insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. At least two dose levels shall be used. The highest dose level shall result in toxic effects (which shall not produce an incidence of fatalities which would prevent a meaningful evaluation) or shall be the highest dose attainable or 5g/kg body weight.
(iii) Route of administration. Acceptable routes of administration include oral, inhalation, admixture with food or water, and IP or IV injection.
(e) Test performance – (1) Treatment and mating. The animals shall be dosed with the test substances 7 days per week over a period of 35 days. After treatment, each male shall be caged with 2 untreated females for a period of 1 week. At the end of 1 week, females shall be separated from males and caged individually. When females give birth, the day of birth, litter size, and sex of progeny shall be recorded. All male progeny should be weaned, and all female progeny should be discarded.
(2) Testing for translocation heterozygosity. When males are sexually mature, testing for translocation heterozygosity shall begin. One of two methods shall be used; the first method involves mating, determining those F
(i) Determination of sterility or semisterility – (A) Conventional method. Females are mated, usually three females for each male, and each female is killed at midpregnancy. Living and dead implantations are counted. Criteria for determining normal and semisterile males are usually established for each new strain because the number of dead implantations varies considerably among strains.
(B) Sequential method. Males to be tested are caged individually with females and the majority of the presumably normal males are identified on the basis of a predetermined size of 1 or 2 litters. Breeding pens are examined daily on weekdays beginning 18 days after pairing. Young are discarded immediately after they are scored. Males that sire a litter whose size is the same as or greater than the minimum set for a translocation-free condition are discarded with their litter. If the litter size is smaller than the predetermined number, a second litter is produced with the same rule applying. Males that cannot be classified as normal after production of a second litter are tested further by the conventional method or by cytological confirmation of translocation.
(ii) Cytological analysis. For cytological analysis of suspected semisteriles, the air-drying technique is used. Observation of at least 2 diakinesis-metaphase 1 cells with mutivalent association constitutes the required evidence for the presence of a translocation. Sterile males are examined by one of two methods, those with testes of normal size and sperm in the epididymis are examined by the same techniques used for semisteriles. Animals with small testes are examined by squash preparations or, alternatively, by examination of mitotic metaphase preparations. If squash preparations do not yield diakinesis-metaphase 1 cells, analysis of spermatogonia or bone marrow for the presence of unusually long or short chromosomes should be performed.
(f) Data and report – (1) Treatment of results. (i) Data shall be presented in tabular form and shall include the number of animals at risk, the germ cell stage treated, the number of partial steriles and semisteriles (if the fertility test is used), the number of cytogenetically confirmed translocation heterozygotes (if the fertility test is used, report the number of confirmed steriles and confirmed partial steriles), the translocation rate, and either the standard error of the rate or the upper 95 percent confidence limit on the rate.
(ii) These data shall be presented for both treated and control groups. Historical or concurrent controls shall be specified, as well as the randomization procedure used for concurrent controls.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of heritable translocations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of heritable translocations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the heritable translocation assay indicate that under the test conditions the test substance causes heritable chromosomal damage in the test species.
(ii) Negative results indicate that under the test conditions the test substance does not cause heritable chromosomal damage in the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Species, strain, age, weight and number of animals of each sex in each group.
(ii) Test chemical vehicle, route and schedule of administration, toxicity data.
(iii) Dosing regimen, doses tested and rationale for dosage selection.
(iv) Mating schedule, number of females mated to each male.
(v) The use of historical or concurrent controls.
(vi) Screening procedure including the decision criteria used and the method by which they were determined.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Generoso, W.M., Bishop, J.B., Goslee, D.G., Newell, G.W., Sheu, G-J, von Halle, E. “Heritable translocation test in mice,” Mutation Research, 76:191-215 (1980).
(2) [Reserved]
§ 798.5500 Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”
(a) Purpose. Bacterial DNA damage or repair tests measure DNA damage which is expressed as differential cell killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient strains. These tests do not measure mutagenic events per se. They are used as an indication of the interaction of a chemical with genetic material implying the potential for genotoxicity.
(b) Definition. Test for differential growth inhibition of repair proficient and repair deficient bacteria measure differences in chemically induced cell killing between wild-type strains with full repair capacity and mutant strains deficient in one or more of the enzymes which govern repair of damaged DNA.
(c) Reference substances. These may include, but need not be limited to, chloramphenicol or methyl methanesulfonate.
(d) Test method – (1) Principle. The tests detect agents that interact with cellular DNA to produce growth inhibition or killing. This interaction is recognized by specific cellular repair systems. The assays are based upon the use of paired bacterial strains that differ by the presence of absence of specific DNA repair genes. The response is expressed in the preferential inhibition of growth or the preferential killing of the DNA repair deficient strain since it is incapable of removing certain chemical lesions from its DNA.
(2) Description. Several methods for performing the test have been described. Those described here are:
(i) Tests performed on solid medium (diffusion tests).
(ii) Tests performed in liquid culture (suspension tests).
(3) Strain selection – (i) Designation. At the present time, Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45) pairs are recommended. Other pairs may be utilized when appropriate.
(ii) Preparation and storage. Stock culture preparation and storage, growth requirements, method of strain identification and demonstration of appropriate phenotypic requirements should be performed using good microbiological techniques and should be documented.
(4) Bacterial growth. Good microbiological techniques should be used to grow fresh cultures of bacteria. The phase of growth and cell density should be documented and should be adequate for the experimental design.
(5) Metabolic activation. Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor supplemented postmitochondrial fraction prepared from the livers of rodents treated with enzyme inducing agents. The use of other species, tissues or techniques may also be appropriate.
(6) Control groups – (i) Concurrent controls. Concurrent positive, negative, and vehicle controls should be included in each assay.
(ii) Negative controls. The negative control should show nonpreferential growth inhibition (i.e., should affect both strains equally). Chloramphenicol is an example of a negative control.
(iii) Genotype specific controls. Examples of genotype specific positive controls are methyl methanesulfonate for polA strains and mitomycin C for rec strains.
(iv) Positive controls to ensure the efficacy of the activation system. The positive control reference substance for tests including a metabolic activation system should be selected on the basis of the type of activation system used in the test.
(v) Other positive controls. Other positive control reference substances may be used.
(7) Test chemicals – (i) Vehicle. Test chemicals and positive and negative control reference substances should be dissolved in an appropriate vehicle and then further diluted in vehicle for use in the assay.
(ii) Exposure concentrations. The test should initially be performed over a broad range of concentrations. Among the criteria to be taken into consideration for determining the upper limits of test chemical concentration are cytotoxicity and solubility. Cytotoxicity of the test chemical may be altered in the presence of metabolic activation systems. For freely soluble nontoxic chemicals, the upper test chemical concentration should be determined on a case by case basis. Because results are expressed as diameters of zones of growth inhibition in the diffusion test, it is most important that the amounts of chemical on the disc (or in the wells) are exact replicates. When appropriate, a positive response should be confirmed by testing over a narrow range of concentrations.
(e) Test performance – (1) Diffusion assay – (i) Disc diffusion assays. Disc diffusion assays. may be performed in two ways:
(A) A single strain of bacteria may be added to an agar overlay or spread on the surface of the agar and the test chemical placed on a filter disc on the surface of the agar or;
(B) DNA repair proficient and DNA repair deficient bacteria may be streaked in a line on the surface of the agar of the same plate and a disc saturated with test chemical placed on the surface of the agar in contact with the streaks.
(ii) Well diffusion assays. In well diffusion assays, bacteria may be either added to the agar overlay or spread onto the surface of the agar. A solution of the test chemical is then placed into a well in the agar.
(2) Suspension assays. (i) A bacterial suspension may be exposed to the test chemical and the number of surviving bacteria determined (as colony-forming units) either as a function of time of treatment or as a function of the concentration of test agent.
(ii) Nonturbid suspensions of bacteria may be exposed to serial dilutions of the test agent and a minimal inhibitory concentration for each strain determined, as evidenced by the presence or absence of visible growth after a period of incubation.
(iii) Paired bacterial suspensions (usually with some initial turbidity) may be treated with a single dose of the chemical. Positive results are indicated by a differential inhibition in the rate of increase of turbidity of the paired cultures.
(3) Number of cultures. When using a plate diffusion procedure, at least two independent plates should be used at each dilution. In liquid suspension assays, at least two independent specimens for determination of the number of viable cells should be plated.
(4) Incubation conditions. All plates in a given test should be incubated for the same time period. This incubation period should be for 18 to 24 hrs at 37 °C.
(f) Data and report – (1) Treatment of results – (i) Diffusion assays. Results should be expressed in diameters of zones of growth inhibition in millimeters or as areas derived therefrom as mm
(ii) Liquid suspension assays. (A) Survival data can be presented as dose responses, preferably as percentage of survivors or fractional survival of each strain or as a relative survival (ratio) of the two strains.
(B) Results can also be expressed as the concentrations required to effect a predetermined survival rate (e.g., D
(C) Similarly, results can be expressed as minimal inhibitory concentration or as minimal lethal dose. The former is determined by the absence of visible growth in liquid medium and the latter is determined by plating dilutions onto semisolid media.
(iii) In all tests, concentrations must be given as the final concentrations during the treatment. Raw data, prior to transformation, should be provided. These should include actual quantities measured, e.g., neat numbers. For measurement of diffusion, the diameters of the discs and/or well should be indicated and the measurements should indicate whether the diameter of the discs and/or well was subtracted. Moreover, mention should be made as to whether the test chemical gave a sharp, diffuse, or double-zone of growth inhibition. If it is the latter, the investigator should indicate whether the inner or the outer zone was measured.
(iv) Viability data should be given as the actual plate counts with an indication of the dilution used and the volume plated or as derived titers (cells per ml). Transformed data alone in the absence of experimental data are not acceptable (i.e, ratios, differences, survival fraction).
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related preferential inhibition or killing of the repair deficient strain. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related preferential inhibition or killing of the repair deficient strain or a statistically significant and reproducible positive response at any one of the test points is considered not to interact with the genetic material of the organisms used in assay.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. DNA damage tests in bacteria do not measure DNA repair per se nor do they measure mutations. They measure DNA damage which is expressed as cell killing or growth inhibition. A positive result in a DNA damage test in the absence of a positive result in another system is difficult to evaluate in the absence of a better data base.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information should be reported:
(i) Bacterial strains used.
(ii) Phase of bacterial cell growth at time of use in the assay.
(iii) Media composition.
(iv) Details of both the protocol used to prepare the metabolic activation system and its use in the assay.
(v) Treatment protocol, including doses used and rationale for dose selection, positive and negative controls.
(vi) Method used for determination of degree of cell kill.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test,” Mutation Research, 31:347-364 (1975).
(2) Kada, T., Sadie, Y., Tutikawa, K. “In vitro and host-mediated “rec-assay” procedures for screening chemical mutagens; and phloxine, a mutagenic red dye detected,” Mutation Research, 16:165-174 (1972).
(3) Leifer, Z., Kada, T., Mandel, M., Zeiger, E., Stafford, R., Rosenkranz, H.S. “An evaluation of bacterial DNA repair tests for predicting genotoxicity and carcinogenicity: A report of the U.S. EPA’s Gene-Tox Program,” Mutation Research, 87:211-297 (1981).
(4) Slater, E.E., Anderson, M.D., Rosenkranz, H.S. “Rapid detection of mutagens and carcinogens.” Cancer Research, 31:970-973 (1971).
§ 798.5955 Heritable translocation test in drosophila melanogaster.
(a) Purpose. The heritable translocation test in Drosophila measures the induction of chromosomal translocations in germ cells of insects. Stocks carrying genetic markers on two or more chromosomes are used to follow the assortment of chromosomes in meiosis. The F
(b) Definitions – (1) Chromosome mutations are chromosomal changes resulting from breakage and reunion of chromosomes. Chromosomal mutations are also produced through nondisjunction of chromosomes during cell division.
(2) Reciprocal translocations are chromosomal translocations resulting from reciprocal exchanges between two or more chromosomes.
(3) Heritable translocations are reciprocal translocations transmitted from parent to the succeeding progeny.
(c) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate or N-dimethyl-nitrosamine.
(d) Test method – (1) Principle. The method is based on the principle that balanced reciprocal chromosomal translocations can be induced by chemicals in the germ cells of treated flies and that these translocations are detected in the F
(2) Description. Wild-type males are treated with chemical and bred with females of known genetic markers. The F
(i) Illustrative example. The following example serves to illustrate the method. Males carrying genes for red eye color on chromosomes II and III are bred with females of white eye color carrying alleles for brown (bw) on the second chromosome and scarlet (st) and pink (pp) on the third chromosome. The F
(ii) [Reserved]
(3) Drosophila stocks. Wild-type males and females of the genotype bw:st:pp (white eyes) may be used in the heritable translocation test. Other appropriately marked Drosophila stocks may also be used.
(4) Control groups. (i) Concurrent positive and negative (vehicle) controls should be included in each experiment.
(ii) Negative (vehicle) controls should be included. The size of the negative (vehicle) control group should be determined by the availability of appropriate laboratory historical control data.
(iii) If the historical control data are of sufficient numbers, concurrent controls may not be necessary.
(5) Test chemicals – (i) Vehicle. Test chemicals should be dissolved in water. Compounds which are insoluble in water may be dissolved or suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-60 or 80), and then diluted in water or saline prior to administration. Dimethylsulfoxide should be avoided as a vehicle.
(ii) Dose levels. For the initial assessment of mutagenicity, it may be sufficient to test a single dose of the test substance. This dose should be the maximum tolerated dose or that which produces some indication of toxicity. If the test is being used to verify mutagenic activity, at least two additional exposure levels should be used.
(iii) Route of administration. Exposure may be oral, by injection or by exposure to gases or vapours. Feeding of the test compound may be done in sugar solution. When necessary, substances may be dissolved in 0.7 percent NaCl solution and injected into the thorax or abdomen.
(e) Test performance – (1) P1 mating. (i) In the primary screen of a chemical, it is enough to sample one germ cell stage, either mature sperm or spermatids (for indirect acting mutagens). Other stages may be sampled if needed, i.e., when mature germ cells give a positive result and data from earlier germ cells are needed for the purpose of risk assessment. Thus, the treated males may be mated only once for a period of 3 days to sample sperm or transferred every 2 to 3 days to cover the entire germ cell cycle.
(ii) Mass matings may be performed because the control rate for translocations in the available literature is very low (near 0) and clustered events are extremely rare. Mated females may be aged for 2 weeks in order to recover an enhanced incidence of translocation due to the storage effect. The females are then allowed to lay eggs and F
(2) F
(3) Scoring the F
(4) Number of replicate experiments. Replicate experiments are usually performed for each dose of the compound tested. If a chemical is a potent inducer of translocations, one experiment may be sufficient. Otherwise two or three replicate experiments should be done.
(f) Data and report – (1) Treatment of results. Data should be tabulated to show the number of translocations and the number of fertile F
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of heritable translocations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of heritable translocations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the heritable translocation test in Drosophila indicate that under the test conditions the test substance causes chromosome damage in germ cells of this insect.
(ii) Negative results indicate that under the test conditions the test substance does not cause chromosomal damage in D. melanogaster.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information should be reported:
(i) Drosophila stock used in the assay, age of insects, number of males treated, number of F
(ii) Test chemical vehicle, treatment and mating schedule, exposure levels, toxicity data, dose and route of exposure.
(iii) Positive and negative (vehicle) controls.
(iv) Historical control data, if available.
(v) Number of chromosomes scored.
(vi) Criteria for scoring mutant chromosomes.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Wurgler, F.E., Sobels, F.H., Vogel, E. “Drosophila as assay system for detecting genetic changes,” Handbook of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M., Nichols, W., Ramel, C. (Amsterdam: Elsevier/North Holland Biomedical Press, 1979) pp. 335-374.
(2) [Reserved]
Subpart G – Neurotoxicity
§ 798.6050 Functional observational battery.
(a) Purpose. In the assessment and evaluation of the potential human health effects of substances, it may be necessary to test for neurotoxic effects. Substances that have been observed to cause neurotoxic signs (e.g., convulsions, tremors, ataxia) in other toxicity tests, as well as those having a structural similarity to known neurotoxicants, should be evaluated for neurotoxicity. The functional observational battery is a noninvasive procedure designed to detect gross functional deficits in young adults resulting from exposure to chemicals and to better quantify neurotoxic effects detected in other studies. This battery of tests is not intended to provide a detailed evaluation of neurotoxicity. It is designed to be used in conjunction with neuropathologic evaluation and/or general toxicity testing. Additional functional tests may be necessary to assess completely the neurotoxic potential of a chemical.
(b) Definitions. (1) Neurotoxicity is any adverse effect on the structure or function of the central and/or peripheral nervous system related to exposure to a chemical substance.
(2) A toxic effect is an adverse change in the structure or function of an experimental animal as a result of exposure to a chemical substance.
(c) Principle of the test method. The material is administered by an appropriate route to laboratory rodents. The animals are observed under carefully standardized conditions with sufficient frequency to ensure the detection of behavioral and/or neurologic abnormalities, if present. Various functions that could be affected by neurotoxicants are assessed during each observation period.
(d) Test procedures – (1) Animal selection – (i) Species and strain. The laboratory rat or mouse is recommended. Although information will generally be lacking, whenever possible the choice of species should take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies. The potential for combined studies should also be considered. Standard strains should be used.
(ii) Age. Young adult animals (at least 42 days old for the rat or mouse) shall be used.
(iii) Sex. (A) Equal numbers of animals of each sex are required for each dose level.
(B) The females shall be nulliparous and nonpregnant.
(2) Number of animals. At least eight animals of each sex should be used at each dose level and should be designated for behavioral testing. If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed before the end of the study. Animals shall be randomly assigned to treatment and control groups.
(3) Control groups. (i) A concurrent (“sham” exposure or vehicle) control group is required. Subjects shall be treated in the same way as for an exposure group except that administration of the test substance is omitted.
(ii) Concurrent or historic data from the laboratory performing the testing shall provide evidence of the ability of the procedures used to detect major neurotoxic endpoints such as limb weakness or paralysis (e.g., acrylamide), CNS stimulation (e.g., β, β′-iminodiproprionitrile) autonomatic signs (e.g., physostigmine).
(iii) A satellite group may be treated with the high dose level for the duration of exposure and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate duration, normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g.,
(i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively.
(5) Duration and frequency of exposure. The duration and frequency of exposure will be specified in the test rule.
(6) Route of exposure. The test substance shall be administered by the route specified in the test rule. This route will usually be the one most closely approximating the expected route of human exposure. The exposure potocol shall conform to that outlined in the appropriate acute or subchronic toxicity study guideline under subpart B or subpart C of this part.
(7) Combined protocol. Subjects used for other toxicity studies may be used if none of the requirements of either study are violated by the combination.
(8) Study conduct. (i) All animals in a given study should be observed carefully by trained technicians who are blind with respect to the animals’ treatments. Standard procedures to minimize observer variability shall be followed. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of inter-observer reliability is required. All animals should be observed prior to initiation of exposure. Subsequent observations should be made with sufficent frequency to ensure the detection of behavioral and/or neurologic abnormalities, if present. At minimum, observations at 1 hour, 6 hours, 24 hours, 7 days, and 14 days and monthly thereafter are recommended. In a subchronic study, subsequent to the first exposure all observations should be made before the daily exposure. The animals should be removed from the home cage to a standard arena for observation. Effort should be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables that can affect behavior are sound level, temperature, humidity, lighting, odors, time of day, and environmental distractions. Explicit, operationally defined scales for each function should be used. The development of objective quantitative measures of the observational endpoints specified is encouraged.
(ii) The following is a minimal list of observations that shall be noted:
(A) Any unusual responses with respect to body position, activity level, coordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to, headflicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards.
(C) The presence of:
(1) Convulsions.
(2) Tremors.
(3) Increased levels of lacrimation and/or red-colored tears.
(4) Increased levels of salivation.
(5) Piloerection.
(6) Pupillary dilation or constriction.
(7) Unusual respiration (shallow, labored, dyspneic, gasping, and retching) and/or mouth breathing.
(8) Diarrhea.
(9) Excessive or diminished urination.
(10) Vocalization.
(D) Forelimb/hindlimb grip strength. The procedure described by Meyer et al. (1979), under paragraph (f)(9) of this section is recommended.
(E) Sensory function. A simple assessment of sensory function (vision, audition, pain perception) shall be made. Marshall et al. (1971) under paragraph (f)(8) of this section have described a neurologic exam for this purpose; these procedures are also discussed by Deuel (1977), under paragraph (f)(4) of this section. Irwin (1968) under paragraph (f)(7) of this section described a number of reflex tests intended to detect gross sensory deficits, including the visual placing response, Preyer reflex, and tail pinch. Many procedures have been developed for assessing pain perception (e.g., Ankier, 1974 under paragraph (f)(1) of this section; D’Amour and Smith 1941 under paragraph (f)(3) of this section; Evans 1971 under paragraph (f)(6) of this section).
(e) Data reporting and evaluation. In addition to the reporting requirements specified under 40 CFR part 792 subpart J the final test report must include the following information.
(1) Description of system and test methods. (i) A detailed description of the procedures used to standardize observation, including the arena and operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historic data may be used if all aspects of the experimental protocol are the same, including personnel.
(2) Results. The following information must be arranged by test group dose level.
(i) In tabular form, data for each animal must be provided showing:
(A) Its identification number.
(B) Its body weight and score on each sign at each observation time, the time and cause of death (if appropriate).
(ii) Summary data for each group must include:
(A) The number of animals at the start of the test.
(B) The number of animals showing each observation score at each observation time.
(C) The percentage of animals showing each abnormal sign at each observation time.
(D) The mean and standard deviation for each continuous endpoint at each observation time.
(3) Evaluation of data. The findings of a functional observational battery should be evaluated in the context of preceding and/or concurrent toxicity studies and any correlative histopathological findings. The evaluation shall include the relationship between the doses of the test substance and the presence or absence, incidence and severity, of any neurotoxic effects. The evaluation should include appropriate statistical analyses. Choice of analyses should consider tests appropriate to the experimental design and needed adjustments for multiple comparisons.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Ankier, S.I. “New hot plate tests to quantify antinociceptic and narcotic antagonist activities,” European Journal of Pharmacology, 27: 1-4 (1974).
(2) Coughenour, L.L., McLean, J.R. and Parker, R.B. “A new device for the rapid measurement of impaired motor function in mice,” Pharmacology, Biochemistry and Behavior, 6: 351-353 (1977).
(3) D’Amour, F.E., Smith, D.L. “A method for determining loss of pain sensation,” Journal of Pharmacology and Experimental Therapeutics, 72: 74-79 (1941).
(4) Deuel, R.K. “Determining sensory deficits in animals,” Methods in Psychobiology Ed. Myers R.D. (New York: Academic Press, 1977) pp. 99-125.
(5) Edwards, P.M., Parker, V.H. “A simple, sensitive and objective method for early assessment of acrylamide neuropathy in rats,” Toxicology and Applied Pharmacology, 40: 589-591 (1977).
(6) Evans, W.O. “A new technique for the investigation of some analgesic drugs on reflexive behavior in the rat,” Psychopharmacologia, 2: 318-325 (1961).
(7) Irwin, S. “Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral and physiologic state of the mouse,” Psychopharmacologia, 13: 222-257 (1968).
(8) Marshall, J.F., Turner, B.H., Teitlbaum, P. “Sensory neglect produced by lateral hypothalamic damage,” Science, 174: 523-525 (1971).
(9) Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T. “A method for the routine assessment of fore- and hindlimb grip strength of rats and mice,” Neurobehavioral Toxicology, 1: 233-236 (1979).
§ 798.6200 Motor activity.
(a) Purpose – (1) General. In the assessment and evaluation of the toxic characteristics of a substance, determination of the effects of administration of the substance on motor activity is useful when neurotoxicity is suspected.
(2) Acute Motor Activity Test. The purpose of the acute motor activity test is to examine changes in motor activity occurring over a range of acute exposure levels. These changes may then be evaluated in the context of changes occurring in other organ systems. This test is an initial step in determining the potential of a substance to produce acute neurotoxicity and may be used to screen members of a class of substances for known neurotoxicity, and/or to establish a dosage regimen prior to the initiation of subchronic neurotoxicity testing.
(3) Subchronic Motor Activity Test. The purpose of the subchronic motor activity test is to determine whether the repeated administration of a suspected neurotoxicant results in changes in motor activity. These changes may be evaluated in the context of changes occurring in other organ systems. This test is an initial step in determining the potential of a substance to produce subchronic neurotoxicity.
(b) Definitions. (1) Neurotoxicity is the adverse effect on the structure or function of the central and/or peripheral nervous system related to exposure to a chemical substance.
(2) Motor activity is any movement of the experimental animal.
(3) A toxic effect is an adverse change in the structure or function of an experimental animal as a result of exposure to a chemical substance.
(c) Principle of the test method. The test substance is administered to several groups of experimental animals, one dose being used per group. Measurements of motor activity are made. The exposure levels at which significant changes in motor activity are produced are compared to those levels which produce toxic effects not originating in the central and/or peripheral nervous system.
(d) Test procedures – (1) Animal selection – (i) Species and strain. Testing shall be performed in a laboratory rat or mouse. The choice of species should take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.
(ii) Age. Young adult animals (at least 42 days old for rat or mouse) should be used.
(iii) Sex. (A) Equal numbers of animals of each sex are required for each dose level for the motor activity test.
(B) The females shall be nulliparous and nonpregnant.
(2) Number of animals. Animals shall be randomly assigned to test and control groups. Each test or control group must be designed to contain a sufficient number of animals at the completion of the study to detect a 40 percent change in activity of the test groups relative to the control group with 90 percent power at the 5 percent level. For most designs, calculations can be made according to Dixon and Massey (1957) under paragraph (f)(1) of this section, Neter and Wasserman (1974) under paragraph (f)(5) of this section, Sokal and Rohlf (1969) under paragraph (f)(9) of this section, or Jensen (1972) under paragraph (f)(3) of this section.
(3) Control groups. (i) A concurrent control group is required. This group must be an untreated group, or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control group are required.
(ii) Positive control data are required to demonstrate the sensitivity and reliability of the activity measuring device and testing procedure. These data should demonstrate the ability to detect increases or decreases in activity and to generate a dose-effect curve or its equivalent using three values of the dose or equivalent independent variable. A single administration of the dose (or equivalent) is sufficient. It is recommended that chemical exposure be used to collect positive control data. Positive control data shall be collected at the time of the test study unless the laboratory can demonstrate the adequacy of historical data for this purpose.
(iii) A satellite group may be treated with the high dose level for 90 days and observed for reversibility, persistence or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g.,
(i) The highest dose shall produce (A) clear effects on motor activity or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively.
(5) Duration of testing. The duration of exposure will be specified in the test rule.
(6) Route of administration. The test substance shall be administered by the method specified in the test rule. This will usually be the route most closely approximating the route of human exposure. The exposure protocol shall conform to that outlined in the appropriate acute or subchronic toxicity study guideline.
(7) Combined protocol. The tests described herein may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination.
(8) Study conduct – (i) General. Motor activity must be monitored by an automated activity recording apparatus. The device used must be capable of detecting both increases and decreases in activity, i.e. baseline activity as measured by the device must not be so low as to preclude decreases nor so high as to preclude increases. Each device shall be tested by standard procedure to ensure, to the extent possible, reliability of operation across devices and across days for any one device. In addition, treatment groups must be balanced across devices. Each animal shall be tested individually. The test session shall be long enough for motor activity to approach asymptotic levels by the last 20 percent of the session for most treatments and animals. All sessions should have the same duration. Treatment groups shall be counter-balanced across test times. Effort should be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables which can affect motor activity are sound level, size and shape of the test cage, temperature, relative humidity, lighting conditions, odors, use of home cage or novel test cage and environmental distractions. Tests shall be executed by an appropriately trained individual.
(ii) Acute. Testing shall be timed to include the time of peak signs.
(iii) Subchronic. All animals shall be tested prior to initiation of exposure and at 30 ±2, 60 ±2 and 90 ±2 days during the exposure period. Testing shall occur prior to the daily exposure. Animals shall be weighed on each test day and at least once weekly during the exposure period.
(e) Data reporting and evaluation. In addition to the reporting requirements specified under 40 CFR part 792, subpart J the final test report must include the following information:
(1) Description of system and test methods. (i) Positive control data from the laboratory performing the test which demonstrate the sensitivity of the procedure being used.
(ii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups.
(2) Results. The following information must be arranged by test group (dose level).
(i) In tabular form, data must be provided showing for each animal:
(A) Its identification number.
(B) Body weight, total session activity counts, and intrasession subtotals for each date measured.
(ii) Group summary data should also be reported.
(3) Evaluation of data. An evaluation of the test results (including statistical analysis comparing total activity counts at the end of exposure of treatment vs control animals must be made and supplied. This submission must include dose-effect curves for motor activity expressed as activity counts.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Dixon, W.J., Massey, E.J. Introduction to Statistical Analysis 2nd Ed. (New York: McGraw-Hill, 1957).
(2) Finger, F.W. “Measuring behavioral activity,” Methods in Psychobiology Vol. 2. Ed. R.D. Myers (New York: Academic, 1972) pp. 1-19.
(3) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling’s T
(4) Kinnard, E.J. and Watzman, N. “Techniques utilized in the evaluation of psychotropic drugs on animals activity,” Journal of Pharmaceutical Sciences, 55:995-1012 (1966).
(5) Neter, J. and Wasserman, W. Applied Linear Statistical Models. Homewood, Richard D. Irwin, Inc., 1974.
(6) Reiter, L.E. “Use of activity measures in behavioral toxicology,” Environmental Health Perspectives, 26:9-20 (1978).
(7) Reiter, L.W. and MacPhail, R.C. “Motor Activity: A survey of methods with potential use in toxicity testing,” Neurobehavioral Toxicology, 1: Suppl. 1, 53-66 (1979).
(8) Robbins, T.W. “A critique of the methods available for the measurement of spontaneous motor activity,” Handbook of Psychopharmacology. Vol. 7. Eds. Iversen, L.L., Iversen, D.S., Snyder, S.H. (New York: Plenum, 1977) pp. 37-82.
(9) Sokal, R.P. and Rohlf, E.J. Biometry. (San Francisco: W.H. Freeman and Co., 1969).
§ 798.6400 Neuropathology.
(a) Purpose. The techniques in this guideline are designed to develop data on morphologic changes in the nervous system for chemical substances and mixtures subject to such testing under the Toxic Substances Control Act. The data will detect and characterize morphologic changes, if and when they occur, and determine a no-effect level for such changes. Neuropathological evaluation should be complemented by other neurotoxicity studies, e.g. behavioral and neurophysiological studies. Neuropathological evaluation may be done following acute, subchronic or chronic exposure.
(b) Definition. Neurotoxicity or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical agent.
(c) Principle of the test method. The test substance is administered to several groups of experimental animals, one dose being used per group. The animals are sacrificed and tissues in the nervous system are examined grossly and prepared for microscopic examination. Starting with the highest dosage level, tissues are examined under the light microscope for morphologic changes, until a no effect level is determined. In cases where light microscopy has revealed neuropathology, the no effect level may be confirmed by electron microscopy.
(d) Test procedure – (1) Animal selection – (i) Species and strain. Testing shall be performed in the species being used in other tests for neurotoxicity. This will generally be the laboratory rat. The choice of species shall take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.
(ii) Age. Animals shall be young adults (150-200 gm for rats) at the start of exposure.
(iii) Sex. Both sexes shall be used unless it is demonstrated that one sex is refractory to the effects.
(2) Number of animals. A minimum of six animals per group shall be used. The tissues from each animal shall be examined separately. It is recomse (iv)mended that ten animals per group be used.
(3) Control groups. (i) A concurrent control group(s) is (are) required. This group must be an untreated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the vehicle used has a known or potential toxic property, both untreated and vehicle control groups are required.
(ii) A satellite group of animals may be treated with the high level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length; normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g.,
(i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-dependent effects at two dose levels or (B) no effects at two dose levels, respectively.
(5) Duration of testing. The exposure duration will be specified in the test rule. This will generally be 90 days exposure.
(6) Route of administration. The test substance shall be administered by a route specified in the test rule. This will generally be the route most closely approximating the route of human exposure. The exposure protocol shall conform to that outlined in the appropriate acute or subchronic toxicity guideline.
(7) Combined protocol. The tests described herein may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination.
(8) Study conduct – (i) Observation of animals. All toxicological (e.g., weight loss) and neurological signs (e.g., motor disturbance) shall be recorded frequently enough to observe any abnormality, and not less than weekly.
(ii) Sacrifice of animals – (A) General. The goal of the techniques outlined for sacrifice of animals and preparation of tissues is preservation of tissues morphology to simulate the living state of the cell.
(B) Perfusion technique. Animals shall be perfused in situ by a generally recognized technique. For fixation suitable for light or electronic microscopy, saline solution followed by buffered 2.5 percent glutaraldehyde or buffered 4.0 percent paraformaldehyde, is recommended. While some minor modifications or variations in procedures are used in different laboratories, a detailed and standard procedure for vascular perfusion may be found in the text by Zeman and Innes (1963) under paragraph (f)(7) of this section, Hayat (1970) under paragraph (f)(3) of this section, and by Spencer and Schaumburg (1980) under paragraph (f)(6) of this section. A more sophisticated technique is described by Palay and Chan-Palay (1974) under paragraph (f)(4) of this section.
(C) Removal of brain and cord. After perfusion, the bonystructure (cranium and vertebral column) shall be exposed. Animals shall then be stored in fixative-filled bags at 4 °C for 8-12 hours. The cranium and vertebral column shall be removed carefully by trained technicians without physical damage of the brain and cord. Detailed dissection procedures may be found in the text by Palay and Chan-Palay (1974) under paragraph (f)(4) of this section. After removal, simple measurement of the size (length and width) and weight of the whole brain (cerebrum, cerebellum, pons-medulla) shall be made. Any abnormal coloration or discoloration of the brain and cord shall also be noted and recorded.
(D) Sampling. Unless a given test rule specifies otherwise, cross-sections of the following areas shall be examined: The forebrain, the center of the cerebrum, the midbrain, the cerebellum and pons, and the medulla oblongata; the spinal cord at cervical and lumbar swelling (C
(iii) Specimen storage. Tissue samples from both the central and peripheral nervous system shall be further immersion fixed and stored in appropriate fixative (e.g., 10 percent buffered formalin for light microscopy; 2.5 percent buffered gluteraldehyde or 4.0 percent buffered paraformaldehyde for electron microscopy) for future examination. The volume of fixative versus the volume of tissues in a specimen jar shall be no less than 25:1. All stored tissues shall be washed with buffer for at least 2 hours prior to further tissue processing.
(iv) Histopathology examination. (A) Fixation. Tissue specimens stored in 10 percent buffered formalin may be used for this purpose. All tissues must be immersion fixed in fixative for at least 48 hours prior to further tissue processing.
(B) Dehydration. All tissue specimens shall be washed for at least 1 hour with water or buffer, prior to dehydration. (A longer washing time is needed if the specimens have been stored in fixative for a prolonged period of time.) Dehydration can be performed with increasing concentration of graded ethanols up to absolute alcohol.
(C) Clearing and embedding. After dehydration, tissue specimens shall be cleared with xylene and embedded in paraffin or paraplast. Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded together in one single block for sectioning. All tissue blocks shall be labelled showing at least the experiment number, animal number, and specimens embedded.
(D) Sectioning. Tissue sections, 5 to 6 microns in thickness, shall be prepared from the tissue blocks and mounted on standard glass slides. It is recommended that several additional sections be made from each block at this time for possible future needs for special stainings. All tissue blocks and slides shall be filed and stored in properly labeled files or boxes.
(E) Histopathological techniques. Although the information available for a given chemical substance may dictate test-rule specific changes, the following general testing sequence is proposed for gathering histopathological data:
(1) General staining. A general staining procedure shall be performed on all tissue specimens in the highest treatment group. Hematoxylin and eosin (H&E) shall be used for this purpose. The staining shall be differentiated properly to achieve bluish nuclei with pinkish background.
(2) Special stains. Based on the results of the general staining, selected sites and cellular components shall be further evaluated by the use of specific techniques. If H&E screening does not provide such information, a battery of stains shall be used to assess the following components in all appropriate required samples: neuronal body (e.g., Einarson’s gallocyanin), axon (e.g., Bodian), myelin sheath (e.g., Kluver’s Luxol Fast Blue) and neurofibrils (e.g., Bielchosky). In addition, peripheral nerve fiber teasing shall be used. Detailed staining methodology is available in standard histotechnological manuals such as AFIP (1968) under paragraph (f)(1) of this section, Ralis et al. (1973) under paragraph (f)(5) of this section, and Chang (1979) under paragraph (f)(2) of this section. The nerve fiber teasing technique is discussed in Spencer and Schaumberg (1980) under paragraph (f)(6) of this section. A section of normal tissue shall be included in each staining to assure that adequate staining has occurred. Any changes shall be noted and representative photographs shall be taken. If a lesion(s) is observed, the special techniques shall be repeated in the next lower treatment group until no further lesion is detectable.
(3) Alternative technique. If the anatomical locus of expected neuro-pathology is well-defined, epoxy-embedded sections stained with toluidine blue may be used for small sized tissue samples. This technique obviates the need for special stains for cellular components. Detailed methodology is available in Spencer and Schaumberg (1980) under paragraph (f)(6) of this section.
(4) Electron microscopy. Based on the results of light microscopic evaluation, specific tissue sites which reveal a lesion(s) shall be further evaluated by electron microscopy in the highest treatment group which does not reveal any light microscopic lesion. If a lesion is observed, the next lower treatment group shall be evaluated until no significant lesion is found. Detailed methodology is available in Hayat (1970) under paragraph (f)(3) of this section.
(F) Examination – (1) General. All stained microscopic slides shall be examined with a standard research microscope. Examples of cellular alterations (e.g., neuronal vacuolation, degeneration, and necrosis) and tissue changes (e.g., gliosis, leukocytic infiltration, and cystic formation) shall be recorded and photographed.
(2) Electron microscopy. Since the size of the tissue samples that can be examined is very small, at least 3 to 4 tissue blocks from each sampling site must be examined. Tissue sections must be examined with a transmission electron microscope. Three main categories of structural changes must be considered:
(i) Neuronal body. The shape and position of the nucleus and nucleolus as well as any change in the chromatin patterns shall be noted. Within the neuronal cytoplasm, cytoplasmic organelles such as mitochondria, lysosomes, neurotubules, neurofilaments, microfilaments, endoplasmic reticulum and polyribosomes (Nissl substance), Golgi complex, and secretory granules shall be examined.
(ii) Neuronal processes. The structural integrity or alterations of dendrites, axons (myelinated and unmyelinated), myelin sheaths, and synapses shall be noted.
(iii) Supporting cells. Attention must also be paid to the number and structural integrity of the neuroglial elements (oligodendrocytes, astrocytes, and microglia) of the central nervous system, and the Schwann cells, satellite cells, and capsule cells of the peripheral nervous system. Any changes in the endothelial cells and ependymal lining cells shall also be noted whenever possible. The nature, severity, and frequency of each type of lesion in each specimen must be recorded. Representative lesions must be photographed and labeled appropriately.
(e) Data collection, reporting, and evaluation. In addition to information meeting the requirements stated under 40 CFR part 792 subpart J, the following specific information shall be reported:
(1) Description of test system and test methods. A description of the general design of the experiment shall be provided. This shall include a short justification explaining any decisions where professional judgment is involved such as fixation technique and choice of stains.
(2) Results. All observations shall be recorded and arranged by test groups. This data may be presented in the following recommended format:
(i) Description of signs and lesions for each animal. For each animal, data must be submitted showing its identification (animal number, treatment, dose, duration), neurologic signs, location(s) nature of, frequency, and severity of lesion(s). A commonly-used scale such as 1 + , 2 + , 3 + , and 4 + for degree of severity ranging from very slight to extensive may be used. Any diagnoses derived from neurologic signs and lesions including naturally occurring diseases or conditions, should also be recorded.
(ii) Counts and incidence of lesions, by test group. Data shall be tabulated to show:
(A) The number of animals used in each group, the number of animals displaying specific neurologic signs, and the number of animals in which any lesion was found;
(B) The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesion.
(iii) Evaluation of data. (A) An evaluation of the data based on gross necropsy findings and microscopic pathology observations shall be made and supplied. The evaluation shall include the relationship, if any, between the animal’s exposure to the test substance and the frequency and severity of the lesions observed.
(B) The evaluation of dose-response, if existent, for various groups shall be given, and a description of statistical method must be presented. The evaluation of neuropathology data should include, where applicable, an assessment in conjunction with other neurotoxicity studies performed (eg. electrophysiological, behavioral, neurochemical).
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) AFIP. Manual of Histologic Staining Methods. (New York: McGraw-Hill (1968).
(2) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry. (Springfield, IL: Charles C. Thomas, 1979).
(3) Hayat, M.A. “Vol. 1. Biological applications,” Principles and techniques of electron microscopy. (New York: Van Nostrand Reinhold, 1970)
(4) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and Organization. (New York: Springer-Verlag, 1974).
(5) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in Neurohistology. (London: Butterworths, 1973).
(6) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).
(7) Zeman, W., JRM Innes, J.R.M. Craigie’s Neuroanatomy of the Rat. (New York: Academic, 1963).
§ 798.6500 Schedule-controlled operant behavior.
(a) Purpose. (1) In the assessment and evaluation of the potential human health effects of substances, it may be necessary to test for functional neurotoxic effects. Substances that have been observed to produce neurotoxic signs in other toxicity studies (e.g. CNS depression or stimulation), as well as substances with a structural similarity to known neurotoxicants should be evaluated for these effects.
(2) This guideline defines procedures for conducting studies of schedule-controlled operant behavior, one way of evaluating functional neurotoxic effects (Dews, 1972 under paragraph (f)(1) of this section; NAS 1975, 1977, 1982 under paragraphs (f)(4), (5) and (6) of this section). Our purpose is to evaluate the effects of acute and repeated exposures on the rate and pattern of responding under schedules of reinforcement. Operant behavior tests may be used to evaluate many other aspects of behavior (Laties, 1978 under paragraph (f)(3) of this section). Additional tests may be necessary to completely assess the behavioral effects of any substance. Behavioral evaluation should be used in conjunction with neuropathologic evaluation and the evaluation of other toxic effects.
(b) Definitions – (1) Neurotoxicity. Neurotoxicity or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical agent. Behavioral toxicity is an adverse change in the functioning of the organism with respect to its environment following exposure to a chemical agent.
(2) Operant, operant behavior, operan