43820 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

ENVIRONMENTAL PROTECTION standard can modify these guidelines as Agency experts and, in some instances, AGENCY needed for an individual test substance. presented at domestic and international The Agency uses a system where colloquia to solicit the views of 40 CFR Part 799 standardized guidelines are organized recognized experts and the regulated by testing endpoint and codified in a community. The draft harmonized [OPPTS±42193; FRL±5719±5] subpart of this part. When a test rule is guidelines are made available as public RIN 2070±AB76 promulgated, the test standard specified drafts. A notice is published in the in the test rule cross-references the Federal Register announcing their Toxic Substances Control Act Test guideline for the bulk of the testing availability and soliciting public Guidelines requirements. In this context, the public comment. is given notice of, and an opportunity to Seven of the 11 health effects test AGENCY: Environmental Protection comment on, the guidelines as they are guidelines that are being codified in Agency (EPA). applied in chemical-specific test rules. subpart H of 40 CFR part 799 have their ACTION: Final rule. This approach eliminates the need to origin in this harmonization process. A repeat the same test specifications for notice was published in the Federal SUMMARY: This rule establishes 11 Toxic each substance-specific test rule since Register of June 20, 1996, (61 FR 31522 Substances Control Act (TSCA) health most of the specifications for testing do (FRL–5367–7)) announcing the effects test guidelines in the Code of not change across substances. The test availability of the proposed test Federal Regulations (CFR). specifications in a guideline can be guidelines for Series 870—Health Establishment of these guidelines is varied, when necessary, to the specific Effects Test Guidelines and soliciting necessary to ensure enforceable test requirements of a test rule by language public comment. Comments were standards in test rules promulgated in the test rule itself. received, and a meeting of the Agency’s under section 4 of TSCA. Codification of In 1985, the Agency established a set Federal Insecticide, Fungicide, and this series of TSCA test guidelines does of TSCA test guidelines in 40 CFR parts Rodenticide Act (FIFRA) Scientific not by itself impose obligations upon 795 through 798 (50 FR 39252, Advisory Panel (SAP) was held on any person. Obligations are only September 27, 1985). These guidelines October 29 and 30, 1996. The SAP, an imposed when these guidelines are were established as standardized advisory committee consisting of cross-referenced in a test rule protocols for laboratory testing of an scientific experts both inside and promulgated under section 4 of TSCA. effect or characteristic deemed outside the U.S. Government, reviewed DATES: This rule is effective on August important for the evaluation of health or the guidelines and made comments. The 15, 1997. environmental hazards of a chemical. Agency reviewed these comments in developing the harmonized health FOR FURTHER INFORMATION CONTACT: Standardized guidelines are necessary effects guidelines. Susan Hazen, Director, Environmental for the establishment of enforceable test Four of the 11 guidelines Assistance Division (7408), Office of standards in test rules promulgated (§ § 799.9510, 799.9530, 799.9538, and Pollution Prevention and Toxics, under section 4 of TSCA. 799.9539) were initially developed by Environmental Protection Agency, Rm. The Agency has over time amended the OECD. E-543B, 401 M St., SW., Washington, DC and improved these guidelines (52 FR 20460; telephone: (202) 554–1404; TDD: 19072, May 20, 1987). In order to reduce III. TSCA Test Guidelines (202) 554–0551; e-mail: TSCA- the text of the CFR, the Agency deleted those guidelines which had not been Harmonization has resulted in [email protected]. For specific significantly improved guidelines. information regarding this action or cited in any test rules (60 FR 31917, June 19, 1995 (FRL–4955–2)). However, creating a single set of related activities, please contact Roger guidelines which can be used by both Nelson, Chemical Control Division, II. OPPTS Harmonized Test Guidelines OPP, in its administration of the FIFRA OPPT; telephone: (202) 260–8163; e- EPA is undertaking a comprehensive and the Federal Food, Drug and mail: [email protected]. modification, or harmonization, of its Cosmetic Act (FFDCA), and the Office of SUPPLEMENTARY INFORMATION: This final pesticides and toxics guidelines for Pollution Prevention and Toxics rule establishes a new series of TSCA testing of health effects, environmental (OPPT), which administers TSCA test guidelines in the CFR. effects, and chemical fate. The rationale presented certain challenges. Under FIFRA, test guidelines are used I. Introduction for this harmonization is to incorporate state of the art science, and to minimize in an interactive process between the Section 4(b)(1)(B) of TSCA requires variations among the protocols Agency and registrants seeking that test rules promulgated under the contained in: registration of pesticides or food residue authority of TSCA section 4 include 1. Test guidelines developed by the tolerances. Flexibility to tailor required ‘‘standards for the development of test EPA Office of Pesticide Programs (OPP), testing to individual circumstances is data for such substance or mixture * * which appeared in publications of the critical, and the Agency has *.’’ Test rules promulgated under TSCA National Technical Information Service. considerable discretion to determine section 4 must specify the standards for 2. The series of TSCA test guidelines whether submitted test results are the development of data. Standards established in 1985, which are adequate to support the requested established in test rules for the contained in 40 CFR parts 795, 796, 797, action. Under this scheme, registrants development of data must specify how and 798. have an intrinsic motivation to conduct the study is to be conducted, what data 3. Guidelines published by the well-grounded testing. Thus, pesticide will be collected, and how the data will Organization for Economic Cooperation testing protocols tend to have few be analyzed. The Agency has found that and Development (OECD). absolute requirements specifying the these specifications to a large degree can Harmonization operates as follows: details of the conduct of the testing. be standardized into a common set of EPA scientists develop guidelines (or By contrast the Agency is required protocols, or, as the Agency terms them, modify existing guidelines) for specific under section 4 of TSCA to impose ‘‘guidelines.’’ These guidelines are endpoints. The new or rewritten prescriptive test requirements by notice organized by testing endpoint. Each test guidelines are reviewed by other and comment rulemaking. Rules Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43821 promulgated under section 4 of TSCA older, 1985 non-harmonized guidelines essentially those resulting from the specify classes of affected parties, in 40 CFR parts 795 through 798. The harmonization process with minor usually manufacturers and processors of only significant difference between the changes to promote enforceability. EPA the chemical being specified for testing, TSCA test guidelines and the OPPTS has elected to codify these new rather than interacting with companies harmonized test guidelines is that guidelines in part 799 so as to on an individual basis. These certain recommended procedures in the distinguish them from the pre- rulemakings typically take years to OPPTS harmonized test guidelines are harmonization guidelines in 40 CFR complete. Without initiating another made mandatory (i.e., the guideline parts 795 through 798. rulemaking process, the Agency has the states that they ‘‘shall’’ be carried out). These guidelines will be placed in a ability to require further testing only if IV. Codification in 40 CFR Part 799 new subpart H of part 799. In addition, the tests were not conducted in EPA plans to reserve additional subparts The Agency had originally planned accordance with the procedures of part 799 for test guidelines, so that not to publish the guidelines in the CFR, specified in the test rule. In addition, the structure of part 799 would be as but to instead make the guidelines the Agency has an alternative process of follows: negotiating TSCA testing requirements available via other means (such as the via enforceable consent agreements Internet) and reference the guidelines in Subpart A—General Provisions (ECAs), but these agreements require the specific test rules using the Subpart B—Specific Chemical Test consent of all the parties involved. incorporation by reference procedures Rules provided by 5 U.S.C. 552(a)(1)(E) and 1 Under TSCA section 4 enforceable Subpart C—Testing Consent Orders test standards, much in the conduct of CFR part 51. In the Federal Register these test protocols is left to the document proposing the TSCA section 4 Subpart D—Multichemical Test Rules judgment of those professionals test rule for 21 hazardous air pollutant Subpart E—G [Reserved] conducting the testing. EPA believes substances (HAPs) (61 FR 33178, 33187, Subpart H—Health Effects Test that certain provisions must be June 26, 1996 (FRL–4869–1)), the Guidelines mandatory whenever the guidelines are Agency stated that it was considering cross-referenced in specific test rules. using incorporation by reference. The TSCA test guidelines currently in Therefore, the Agency has used the Subsequently, however, the Director of 40 CFR parts 795 through 798 will be OPPTS harmonized health effects test the Office of Federal Register advised retained for so long as there exist test guidelines developed using the public EPA that the planned TSCA section 4 rules whose data reimbursement periods notice and comment process described process for guideline incorporation was under TSCA section 4(c) have not in Unit II. of this preamble as well as not eligible for incorporation by expired and which cross-reference the certain OECD guidelines to create the reference under 1 CFR part 51. guidelines. TSCA-specific test guidelines which are Therefore, the Agency finds it necessary This table identifies the TSCA test the subject of this rule. Future TSCA to codify a separate set of TSCA test guideline number with its comparable section 4 test rules will cross-reference guidelines into the CFR. As discussed in OPPTS harmonized test guideline part 799 guidelines rather than the this preamble, the TSCA guidelines are public draft.

TABLE 1.ÐTSCA Test Guidelines Cross-Referenced to the OPPTS Harmonized Test Guidelines

OPPTS harmonized test guide- Guideline title TSCA 40 CFR section line (public draft)

TSCA acute inhalation toxicity with histopathology ...... 799.9135 ...... 870.1350 TSCA subchronic inhalation toxicity ...... 799.9346 ...... 870.3465 TSCA prenatal developmental toxicity ...... 799.9370 ...... 870.3700 TSCA reproduction and fertility effects ...... 799.9380 ...... 870.3800 TSCA carcinogenicity ...... 799.9420 ...... 870.4200 TSCA bacterial reverse mutation test ...... 1799.9510 ...... 1OECD 471 and 472 TSCA in vitro mammalian cell gene mutation test ...... 1799.9530 ...... 1OECD 476 TSCA mammalian bone marrow chromosomal aberration test ...... 1799.9538 ...... 1OECD 475 TSCA mammalian erythrocyte micronucleus test ...... 1799.9539 ...... 1OECD 474 TSCA neurotoxicity screening battery ...... 799.9620 ...... 870.6200 TSCA immunotoxicity ...... 799.9780 ...... 870.7800 1The four TSCA genetic toxicity testing guidelines were adopted from the OECD guideline series and not the OPPTS public drafts.

Codification of these guidelines does test standards and any modifications A. Section 799.9135 TSCA Acute not itself impose any obligations on any thereto will be subject to public notice Inhalation Toxicity with Histopathology person. Obligations are imposed only and comment. 1. EPA dropped the requirement for a when the guidelines are cross- V. Guideline by Guideline Discussion 1-hour (hr) exposure test. The Agency referenced in individual TSCA section 4 recognizes that such a technically rulemakings. When cross-referenced in In this unit is a summary of the difficult test would not be likely to yield such test rules, the pertinent TSCA significant changes made to the 11 useful information due to complicating guidelines serve as test standards for harmonized guidelines proposed on factors such as biological rhythms and only these particular section 4 rules. June 20, 1996, which are being inapplicability to insoluble or EPA may propose modifications to the published in this document. chemically inactive particulates. various guidelines as they are utilized Instead, EPA is requiring a 4-hr for chemical-specific test rules. In each exposure point with a trigger for an 8- chemical-specific test rule, the proposed hr exposure point. Test sponsors have 43822 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations the option to extrapolate from shorter- C. Section 799.9370 TSCA Prenatal and capsule administration. This change term exposures. Developmental Toxicity was made to eliminate the disparity 2. EPA dropped the requirement for EPA made no significant changes to between the original 7-day specification performing histopathology in all this guideline. for capsules and 5 days for gavage since animals and substituted a triggered there was no justification for this approach (wherein gross pathology will D. Section 799.9380 TSCA Reproduction disparity. be performed only when the frequency and Fertility Effects 2. EPA changed the terms used for and severity of adverse effects for dosed 1. EPA added the requirement for a certain weekly observations from animals are greater than those for triggered quantitative evaluation of ‘‘motor activity’’ to ‘‘level of activity’’ control animals in the study). primordial follicles from qualitative and from ‘‘grip strength’’ to ‘‘altered 3. EPA dropped the requirement of a evidence of a possible treatment-related strength’’ to reinforce the point that breathing zone purity determination as effect. While the Agency recognizes that these observations need not be unnecessary since the Agency now there are issues concerning the validity automated. believes that standard inhalation of existing methods used to screen 3. The requirement for the toxicology will provide the purity ovarian-primordial follicle counts, the immunotoxicity screen has been measurement of the test substances. Agency believes that the necessity to deleted. The Agency agreed that the 4. EPA requires only a single control identify early senescence in females immunotoxicity screen conducted at group in some circumstances. If both 4- outweighs these concerns. EPA study termination would provide little and 8-hr exposures are being conducted considers data about the effects of meaningful information on the potential in the study, then there would be a chemical substances on effects such as toxicity of the chemical on the immune single control at the 8-hr exposure early female senescence to be essential function system due to the geriatric provided adequate historical control to protecting human health. changes in the animals. data show no changes in histopathology 2. EPA reduced the requirement for 4. EPA deleted the requirement for the or bronchoalveolar lavage between taking organ weights for pups already weighing of spleens because their controls for these test periods. If the 8- opened for necropsy. The guideline only weight would be unacceptably variable hr exposure is being performed as a requires organ weight data from one due to the amount of blood lost during result of the 4-hr trigger, there would randomly selected pup/sex/litter rather the exsanguination process. (The need to be control groups for both 4- than the three pups specified in the weighing of spleens is still a and 8-hr exposure groups. public draft. The Agency believes that requirement in the immunotoxicity 5. EPA redefined the test exposure to collection of organ weight data from one guideline). 4 hrs of exposure to the target pup/sex/litter rather than three will concentration as defined by an average reduce burdens without compromising F. Genetic Toxicity Testing of plus or minus 5% for gases and plus the ability to detect a treatment-related or minus 11% for particles. This effect on brain, spleen, or thymus 1. Section 799.9510 TSCA Bacterial redefinition establishes exposure weight. The random selection is to be Reverse Mutation Test. tolerances, which better assures known made from the population of pups 2. Section 799.9530 TSCA In Vitro test concentration than the original already opened for necropsy. Mammalian Cell Gene Mutation Test. provision which only allowed for test 3. EPA reduced the requirement that exposure after the test chamber reached 20 adult animals per sex per exposure 3. Section 799.9538 TSCA equilibrium. group be examined for histopathology to Mammalian Bone Marrow 6. EPA now distinguishes air change 10 animals (randomly chosen) per sex Chromosomal Aberration Test. requirements between nose-only per exposure group. This reduction was exposure (300 milliter (mL)/minutes made because there would be little 4. Section 799.9539 TSCA (min)/animal) and whole-body exposure additional statistical value in examining Mammalian Erythrocyte Micronucleus (at least 12 to 15 air changes per hr). more than 10 animals per sex per group. Test. 7. EPA changed its description of the Since the guideline still requires that EPA is incorporating these genetic respiratory histopathology requirements gross necropsy and organ weight data be toxicity guidelines directly from the to ensure that inflated state and fixed collected for all parental animals and OECD versions. The Agency made pressure with infusion fixation are used that the weighed organs be preserved, format changes in order to ensure to prepare the lungs for examination. questions about interpretation of consistency with the TSCA test 8. EPA added the requirement to marginal histopathological effects can guidelines format. The Agency actively specify the anatomical location where be resolved by evaluation of the tissues participated in international discussions the four sections are to be taken for from these animals. regarding the development of these nasal histopathology. 4. EPA dropped the requirement of guidelines. EPA participated in the review of the OECD drafts. EPA believes B. Section 799.9346 TSCA Subchronic histopathology of developmental that because these OECD guidelines Inhalation Toxicity anomalies observed macroscopically in F1 and F2 weanlings. Since the intent were developed with international 1. EPA changed the terms used for of this requirement was to confirm the scientific input through the OECD certain weekly observations from nature of the lesions already identified guideline development process, they ‘‘motor activity’’ to ‘‘level of activity’’ macroscopically, the Agency believes provide state-of-the-art guidance which and from ‘‘grip strength’’ to ‘‘altered that the added value of the information is equivalent to and more broadly strength’’ to reinforce the point that would not be worth the cost of the accepted than that in the OPPTS these observations need not be evaluation. harmonized test guidelines public drafts automated. published on June 20, 1996. The process 2. ‘‘Dose’’ and ‘‘dose level’’ were E. Section 799.9420 TSCA EPA used in developing the four TSCA changed to ‘‘concentration’’ and Carcinogenicity genetic toxicity test guidelines is ‘‘dosing’’ was changed to ‘‘exposure’’ to 1. EPA revised the guideline to allow described in reference 5 of Unit VI. of reflect that this is an inhalation study. 5-day per week dosing for both gavage this preamble. Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43823

G. Section 799.9620 TSCA Neurotoxicity rulemaking under docket control action is not considered under Screening Battery number OPPTS–42193 (including Executive Order 12898 (59 FR 7629, EPA made no significant changes to comments and data submitted February 16, 1994) as having a the public draft of this guideline electronically). This record contains the disproportionately high and adverse although EPA made two clarifications to basic information considered by EPA in human health or environmental effect address SAP concerns. Clarifications to developing this rule. EPA will on minority populations and low- the positive control treatment were supplement this record as necessary. income populations. In addition, the made to indicate that such testing need A public version of this record, action is not subject to Executive Order not be done as frequently as every 12 including printed, paper versions of 13045 ‘‘Protection of Children From months. Examples were eliminated to electronic comments, which does not Environmental Health Risks and Safety clarify EPA’s position that permanently include any information claimed as Risk’’ (62 FR 19885, April 23, 1997) injurious chemicals are not necessary, Confidential Business Information (CBI), since it is neither economically though EPA continues to believe that is available for inspection from 12 noon significant under Executive Order 12866 chemical exposures are appropriate to 4 p.m., Monday through Friday, nor does it concern an environmental except legal holidays. The public record health risk or safety risk that an agency H. Section 799.9780 TSCA is located in the TSCA Nonconfidential has reason to believe may Immunotoxicity Information Center, Rm. NE-B607, 401 disproportionately affect children. M St., SW., Washington, DC 20460. 1. EPA incorporated the C. Paperwork Reduction Act recommendation of the SAP that the The record includes the following requirement for flow cytometric analysis information: This rule does not contain of lymphocyte and Natural Killer (NK) 1. Public drafts of seven OPPTS information collection requirements that cell phenotypes be eliminated. A test for harmonized health effects guidelines. necessitate the approval of OMB under the primary antibody (IgM) response to 2. Four OECD genetic toxicity test the Paperwork Reduction Act of 1980 sheep red blood cell (PFC) or enzyme guidelines. (44 U.S.C. 3501 et seq.). linked immunosorbent assay (ELISA) 3. References contained in TSCA would still be required. The guideline health effects test guidelines D. Regulatory Flexibility Act now sets the required exposure time for promulgated in this document. 4. Final report of the FIFRA Scientific The guidelines codified in this the anti-sheep red blood cells (SRBC) document do not constitute a rule for assay at 28 days, thus providing Advisory Panel meeting, held October 29–30, 1996. which EPA must publish a general information on the effects of the test notice of proposed rulemaking under 5 material on non-specific immunity. 5. USEPA. Memorandum, Angela Auletta to Roger Nelson. HAPs Rule: U.S.C. 553(b). Therefore, sections 603 2. EPA adopted the SAP and 604 of the Regulatory Flexibility recommendation to delete the ‘‘optional OECD Process for Update of Genetic Toxicity Test Guidelines. March 10, Act, 5 U.S.C. 603 and 604 do not apply immunotoxicity screen’’ because to this action. lymphocyte phenotyping by flow 1997. cytometry should be an option. VII. Regulatory Assessment E. Unfunded Mandates Reform Act 3. EPA added the requirement that Requirements appropriate species-specific monoclonal Title II of the Unfunded Mandates antibodies be used in the phenotyping A. Waiver of Notice of Proposed Reform Act of 1995 (UMRA), Pub. L. assay. The Agency accepts the SAP Rulemaking and Delay in Effective Date 104–4, which establishes requirements recommendation that this will allow Because the test guidelines codified in for Federal agencies to assess the effects sufficient flexibility to allow for future this document have no substantive of certain regulatory actions on State, advances in flow cytometry and effect on any person without further local, and tribal governments and the antibody marker technology. rulemaking, and such rulemaking would private sector, does not apply. This 4. EPA adopted the SAP be conducted under public notice and action contains neither a private sector recommendation that a minimum of comment procedures, EPA finds that nor an intergovernmental mandate eight animals per treatment group be public notice and comment are because it does not impose an used in order to yield a sufficient unnecessary for this action. Thus, this enforceable duty on anyone. statistical power to detect a 20% change rule may be promulgated without prior Furthermore, a written statement is not based upon the inter-animal variation opportunity for public notice and required under section 202 of UMRA usually encountered in these assays. comment, pursuant to the because section 202 only applies to 5. EPA added the intraperitoneal Administrative Procedure Act, 5 U.S.C. rules for which a general notice of route of exposure to the guideline in 553(b)(3)(B), and may be made effective proposed rulemaking was published, response to the SAP comment that this immediately, without a 30-day delay, and no such notice was issued for this is an acceptable method for pursuant to 5 U.S.C. 553(d)(3). rule. immunization with SRBCs. B. Executive Order 12866, Executive F. Submission to Congress and the 6. EPA adopted the SAP Order 12898, and Executive Order General Accounting Office recommendation that testing 13045 laboratories need not perform a positive This action is not a major rule as control after every experiment. Instead, This action is not subject to Executive defined by 5 U.S.C. 804(2). Pursuant to it is sufficient to include this control Order 12866 (58 FR 51735, October 4, 5 U.S.C. 801(a)(1)(A), EPA has every 6 months or whenever new 1993) since, as explained in Units I. and submitted a report containing this rule reagents are titrated. IV. of this preamble, the guidelines are and other required information to the not intended to have the force and effect U.S. Senate, the U.S. House of VI. Public Record of law until they are cross-referenced in Representatives, and the Comptroller The official record for this future test rules through public notice General of the General Accounting rulemaking, as well as the public and comment procedures that establish Office prior to its publication in today’s version, has been established for this those rules. For the same reason, this Federal Register. 43824 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

List of Subjects in 40 CFR Part 799 response relationship for sensitive the measured toxic response, whether ± Environmental protection, Chemicals, endpoints following acute exposure and expressed as a group mean ( standard Hazardous substances, Health, to characterize toxicologic response deviation) in the case of a continuous Reporting and recordkeeping following acute high exposures. The variable or as incidence in the case of requirements. latter is of particular concern in relation a quantal variable. This definiton to spills and other accidental releases. should not preclude the exploration of Dated: August 7, 1997. This testing is designed to determine the other dose metrics in establishing this Lynn R. Goldman, gross pathology and histopathology relationship. resulting from acute inhalation exposure Geometric standard deviation (GSD) Assistant Administrator for Prevention, to a substance. Because toxic effects on is a dimensionless number equal to the Pesticides and Toxic Substances. the respiratory tract are of particular ratio between the mass median Therefore, 40 CFR part 799 is concern following inhalation exposure, aerodynamic diameter (MMAD) and amended as follows: several indicators of respiratory toxicity either 84% or 16% of the diameter size consisting of histopathology on fixed distribution (e.g., MMAD = 2 µm; 84% PART 799Ð[AMENDED] tissue and evaluation of cellular and = 4 µm; GSD = 4/2 = 2.0.) The MMAD, 1. The authority citation for part 799 biochemical parameters in together with the GSD, describe the continues to read as follows: bronchoalveolar lavage fluid should be particle size distribution of an aerosol. Authority: 15 U.S.C. 2603, 2611, 2625. employed. The respiratory Use of the GSD may not be valid for histopathology consists of specialized non-lognormally distributed aerosols. (If 2. By adding a new paragraph (d) to techniques to preserve tissues of the the size distribution deviates from the § 799.1 to read as follows: respiratory tract in order to allow lognormal, it shall be noted). detailed microscopic examination to Inhalability is the ratio of the number § 799.1 Scope and purpose. identify adverse effects of chemical concentration of particles of a certain * * * * * substances on this organ system. The aerodynamic diameter, dae, that are (d) This part contains certain TSCA bronchoalveolar lavage is designed to be inspired through the nose or mouth to test guidelines which are cross- a rapid screening test to provide an the number concentration of the same referenced in the test rules contained in early indicator of pulmonary toxicity by dae present in the inspired volume of this part. examining biochemical and cytologic ambient air. In humans, inhalability can µ 3. By adding and reserving subparts E endpoints of material from the lungs of exceed 15 m dae, whereas inhalability through G. animals exposed to potentially toxic dramatically decreases for particles 4. By adding a new subpart H, chemical substances. These acute tests above 4 µm dae in small laboratory consisting of § § 799.9135–799.9780, to are designed to assess the relationship, animals. read as follows: if any, between the animals’ exposure to Lower respiratory tract consists of the test substance and to demonstrate those structures of the respiratory tract Subpart HÐHealth Effects Test relationship between the animals’ below the larynx. Guidelines exposure and the incidence and severity Mass geometric mean aerodynamic 799.9135 TSCA acute inhalation toxicity of observed abnormalities, including diameter or the mass median with histopathology. gross or histopathologic lesions, body aerodynamic diameter (MMAD) is the 799.9346 TSCA subchronic inhalation weight changes, effects on mortality, calculated aerodynamic diameter that toxicity. and any other toxic effects. These acute divides the particles of an aerosol (a 799.9370 TSCA prenatal developmental tests are not intended to provide a gaseous suspension of fine liquid or toxicity. complete evaluation of the toxicologic solid particles) in half, based on the 799.9380 TSCA reproduction and fertility weight of the particles. By weight, 50% effects. effects of a substance, and additional 799.9420 TSCA carcinogenicity. functional and morphological of the particles will be larger than the 799.9510 TSCA bacterial reverse mutation evaluations may be necessary to assess MMAD and 50% of the particles will be test. completely the potential effects smaller than the MMAD. 799.9530 TSCA in vitro mammalian cell produced by a chemical substance. Particle regional deposition is the gene mutation test. Additional tests may include longer- fraction of inhaled particles that 799.9538 TSCA mammalian bone marrow term exposures, or more in-depth deposits in the specific region of the chromosomal aberration test. evaluation of specific organ systems as respiratory tract. The major mechanisms 799.9539 TSCA mammalian erythrocyte indicated by signs of toxicity following of particle deposition in the respiratory micronucleus test. acute exposure. tract include impaction, sedimentation, 799.9620 TSCA neurotoxicity screening diffusion, interception, and electrostatic battery. (b) Source. This a new section 799.9780 TSCA immunotoxicity. developed by the United States precipitation. The deposition Environmental Protection Agency. mechanism that is dominant for a given Subpart HÐHealth Effects Test (c) Definitions. The following region depends on the respiratory tract Guidelines definitions apply to this section. architecture and ventilation rate of the Aerodynamic diameter (dae) refers to species and the aerosol particle size and § 799.9135 TSCA acute inhalation toxicity the size of particles. It is the diameter distribution. The respiratory tract in with histopathology. of a sphere of unit density that behaves both humans and various experimental (a) Scope. This section is intended to aerodynamically (has the same settling mammals can be divided into three meet the testing requirements under velocity in air) as the particle of the test regions on the basis of structure, size, section 4 of the Toxic Substances substance. It is used to compare and function: Control Act (TSCA). In the assessment particles of different size, shape, and (1) The extrathoracic region or upper and evaluation of the potential human density, and to predict where in the respiratory tract that includes the nose, health effects of chemical substances, it respiratory tract such particles may be mouth, nasopharynx, oropharynx, is appropriate to test for acute primarily deposited. laryngopharynx, and larynx. inhalation toxic effects. The goals of this Exposure response is the relationship (2) The tracheobronchial region that test are to characterize the exposure- between the exposure concentration and includes the trachea, bronchi, and Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43825 bronchioles (including the terminal laboratory rat and mouse should be exposure-response curve. The bronchioles). used. Under some circumstances, other concentrations can either be linearly or (3) The alveolar region that includes species, such as the hamster or guinea logarithmically spaced depending on the respiratory bronchioles (if present in pig, may be more appropriate, and if the anticipated steepness of the the species), alveolar ducts, alveolar these or other species are used, concentration-response curve. A sacs, and alveoli. justification should be provided. rationale for concentration selection Respiratory effects are any adverse (ii) Strain. If rats and mice are used, should be provided to indicate that the effects on the structure or functions of the use of the F344 rat and the B6C3F1 selected concentrations will maximally the respiratory system related to mouse is preferred to facilitate support detection of concentration- exposure to a chemical substance. comparison with existing data. response relationship. The high Target organ is any organ found to be (iii) Age. Young adults shall be used. concentration should be clearly toxic or a target of toxicity in the 4-hour (hr) The weight variation of animals used in a limit concentration, but should not high concentration group as a result of: a test should not exceed ± 20% of the result in an incidence of fatalities that (1) The initial histopathologic mean weight for each species. would preclude a meaningful evaluation examination (respiratory tract, liver, (iv) Sex. Equal numbers of animals of of the data. The lowest concentration kidney, gross lesions); or each sex shall be used for each should define a no-observed-adverse- (2) The retrospective histopathologic concentration level. The females shall effects level (NOAEL). examination of archived organs be nulliparous and nonpregnant. (i) Limit concentration. For aerosols triggered by their identification as (v) Health status. Body weight and and particles, the high concentrations targets of toxicity in a 90-day study. feed consumption are not sufficient need not be greater than 2 mg/L, or Toxic effects are any adverse changes indicators of the health status of animals concentrations that cannot maintain a (a change that is statistically and prior to initiating an inhalation toxicity particle size distribution having an biologically significant) in the structure study. Prior to initiating the study, MMAD between 1 and 4 µm (i.e., a or function of an experimental animal as animals shall be monitored for known particle size that permits inhalability a result of exposure to a chemical viral and bacterial respiratory pathogens and deposition throughout the substance. determined by conventional respiratory tract). For fibers, the Upper respiratory tract consists of microbiological assays (e.g., serology). bivariate distribution of length and those structures of the respiratory tract The animals shall be free from diameter must ensure inhalability. For above and including the larynx. pathogens at the start of exposure. gases and vapors, the concentrations (d) Principle of the test method. The (2) Number of animals. At least five need not be greater than 50,000 ppm or test substance shall be administered to males and five females shall be used in 50% of the lower explosive limit, several groups of experimental animals; each concentration/duration and control whichever is lower. If a test at an one concentration level and duration group. Animals shall be randomly aerosol or particulate exposure of 2 mg/ being used per group. Bronchoalveolar assigned to treatment and control L (actual concentration of respirable lavage shall be used to evaluate early groups. substance) for 4 hrs or, where this is not effects on the respiratory system by (3) Control groups. The control group feasible, the maximum attainable examining changes in the content of the shall be a sham-treated group. Except concentration, using the procedures lavage fluid of the lung. At 24 hrs for treatment with the test substance, described for this study, produces no following exposure, the animals shall be animals in the control group shall be observable toxic effects, then a full sacrificed and necropsied, and tissue handled in a manner identical to the study using three concentrations will samples from the respiratory tract and test-group animals. Where a vehicle is not be necessary. Similarly, if a test at other major organs will be prepared for used to help generate an appropriate a gas or vapor exposure of 50,000 ppm microscopic examination. The exposure concentration of the substance in the or 50% of the lower explosive limit, levels at which significant toxic effects atmosphere, a vehicle control group whichever is lower, produces no on the respiratory organ system are shall be used. If the 4- and 8-hr observable toxic effects, then a full produced are compared to those levels exposure studies are conducted study using three concentrations will that produce other toxic effects. As concurrently, a concurrent 8-hr sham- not be necessary. triggered by the results of the 4-hr test, exposed control group may serve as the (ii) 8-Hr study and optional 1-hr additional exposure periods of 1 hr and control group for both the 4-hr and the study. If the 8-hr study is triggered, 8 hrs will be required to determine the 8-hr exposure studies, provided there is three concentrations shall be tested. effect of exposure time on the toxicity adequate historical control data showing These concentrations should allow for observed. A 1-hr exposure study can be no changes in histopathology or the determination of an effect level and elected as an option to provide data bronchoalveolar lavage of controls a NOAEL. If the option to perform a 1- suitable for risk assessment for very exposed for 4 and 8 hrs. Similarly, if the hr study is elected, three concentrations short duration exposures as may occur optional 1-hr exposure study is shall be selected and tested in a similar from chemical releases. In the absence conducted concurrently with the 4- and/ manner. of adequate toxicological data for 1-hr or 8-hr study, the concurrent control (5) Inhalation exposure. Animals can exposure, the Agency will extrapolate to group for those studies may also be used be exposed to the substance by either a shorter-term exposures from the 4-hr for the 1-hr study, provided adequate nose-only procedure or in a whole-body data on the basis of concentration alone. historical control data show no changes exposure chamber. This is a conservative method of in histopathology or bronchoalveolar (i) Inhalation chambers. The animals extrapolation, consistent with general lavage between controls exposed for shall be tested in inhalation equipment Agency methods for deriving criteria for these time periods. designed to sustain a dynamic airflow short-term exposure from longer-term (4) Concentration level and for nose-only exposures of at least 300 studies (a concentration x time concentration selection. For the 4-hr ml/minute/animal or an airflow for extrapolation would result in higher study, at least three concentrations shall whole-body exposures of at least 12 to concentration for a shorter duration). be used in addition to the control group. 15 air changes per hr and ensure an (e) Test procedures—(1) Animal Ideally, the data generated from the test adequate oxygen content of at least 19% selection—(i) Species. In general, the should be sufficient to produce an and an evenly distributed exposure 43826 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations atmosphere. Where a whole-body the entire mixture, as well as the levels (rather than only to the extent chamber is used, its design shall principal compound, shall be measured. necessary to define the NOAEL) can minimize crowding by providing (vi) Temperature and humidity shall support the application of exposure- individual caging. As a general rule, to be monitored continuously, but shall be response analyses such as the ensure stability of a chamber recorded at least every 30 minutes. benchmark concentration approach. atmosphere, the total ‘‘volume’’ of the (7) Food and water during exposure (iv) Archived organs identified as test animals should not exceed 5% of period. Food shall be withheld during targets of toxicity from results of the 90- the volume of the test chamber. exposure. Water may also be withheld day study (if a 90-day study is required (ii) Environmental conditions. The in certain cases. for this substance) should be elevated in temperature at which the test is (8) Observation period. The high concentration animals of the 4-hr performed shall be maintained at 22 °C bronchoalveolar lavage and respiratory acute study to determine if they are also ( ±2 °C). Ideally, the relative humidity pathology shall be conducted 24 hrs targets of acute toxicity. should be maintained between 40% and following exposure to allow expression (11) Respiratory tract histopathology. 60%, but in certain instances (e.g., tests of signs of toxicity. There is concern (i) Representative sections of the using water as a vehicle), this may not that some latency time will be required respiratory tract shall be examined be practical. to allow migration of cells and histologically. These shall include the (iii) Exposure periodicity. For acute macromolecules into the lungs trachea, major conducting airways, testing, the exposure design shall enable following exposure, and that some alveolar region, terminal and respiratory 4 hrs of exposure to the target pathology may require macromolecular bronchioles (if present), alveolar ducts concentrations, as defined by an average synthesis or degradation before cell and sacs, and interstitial tissues. of ± 5% for gases and vapors and ± 15% damage develops. (ii) Care shall be taken that the for particles and aerosols. If triggered by (9) Gross pathology. (i) All animals method used to kill the animal does not the results of the 4-hr exposure, shall be subjected to a full gross result in damage to the tissues of the additional testing shall be conducted in necropsy which includes examination upper or lower respiratory tract. The a comparable manner using an 8-hr of orifices and the cranial, thoracic, and lungs shall be infused with a fixative exposure period. abdominal cavities and their contents. while in an inflated state of fixed (6) Physical measurements. (ii) At least the lungs, liver, kidneys, pressure. Measurements or monitoring shall be adrenals, brain, and gonads shall be (iii) The upper respiratory tract shall made of the following: weighed wet, as soon as possible after be examined for histopathologic lesions. (i) Chemical purity of the test material dissection to avoid drying. This examination shall use a minimum shall be analyzed. (iii) The following organs and tissues, of four sections located as specified (ii) The rate of airflow shall be or representative samples thereof, shall under paragraphs (e)(11)(iii)(A) through monitored continuously, but shall be be preserved in a suitable medium for (e)(11)(iii)(D) of this section. An recorded at least every 30 minutes. possible future histopathological evaluation of the nasal vestibule shall be (iii) The actual concentrations of the examination: All gross lesions; brain- conducted. The method described by test substance shall be measured in the including sections of medulla/pons; the reference under paragraph (h)(11) of breathing zone. During the exposure cerebellar cortex and cerebral cortex; this section should be given period, the actual concentrations of the pituitary; thyroid/parathyroid; thymus; consideration. The use of additional test substance shall be held as constant heart; sternum with bone marrow; sections shall be left to the discretion of as practical, monitored continuously or salivary glands; liver; spleen; kidneys; the study pathologist, but consideration intermittently depending on the method adrenals; pancreas; gonads; accessory should be given to additional sections as of analysis, and recorded at least at the genital organs (epididymis, prostrate, recommended in the reference under beginning, at an intermediate time, and and, if present, seminal vesicles); aorta; paragraph (h)(8) of this section to ensure at the end of the exposure period. Well- skin; gall bladder (if present); adequate evaluation of the entire upper established and published monitoring esophagus; stomach; duodenum; respiratory tract, particularly the methods should be used where jejunum; ileum; cecum; colon; rectum; nasopharyngeal meatus. The following available. If no standard methods are urinary bladder; representative lymph transverse sections shall be examined: available, then accuracy and precision nodes; thigh musculature; peripheral (A) Immediately posterior to the information must be supplied. nerve; spinal cord at three levels upper incisor teeth. (iv) During the development of the cervical, midthoracic, and lumbar; and (B) At the incisor papilla. generating system, appropriate particle eyes. Respiratory tract tissues shall also (C) At the second palatal ridge. size analysis shall be performed to be preserved in a suitable medium. (D) At the level of the first upper establish the stability of the aerosol. (10) Histopathology. The following molar teeth. During exposure, analysis should be histopathology shall be performed: (iv) The laryngeal mucosa shall be conducted as often as necessary to (i) Full histopathology shall be examined for histopathologic changes. determine the consistency of particle performed on the respiratory tract, liver Sections of the larynx to be examined size distribution. The particle size and kidney of all animals in the control include the epithelium covering the distribution shall have an MMAD and high concentration groups. The base of the epiglottis, the ventral pouch, between 1 and 4 µm. The particle size histopathology of the respiratory tract is and the medial surfaces of the vocal of hygroscopic materials shall be small described under paragraph (e)(11) of processes of the arytenoid cartilages. enough when dry to assure that the size this section. (12) Bronchoalveolar lavage. (i) of the particle at saturation will still (ii) All gross lesions which differ from Animals can be exposed to the have an MMAD between 1 and 4 µm. controls in frequency, distribution, type, substance by either a nose-only Characterization for fibers shall include or severity in all concentration groups. procedure or in a whole-body exposure the bivariate distribution of length and (iii) Target organs in all animals, as chamber. diameter; this distribution must ensure indicated by the observations in the (ii) Care should be taken that the inhalability. high concentration group in this study. method used to kill the animal results (v) If the test substance is present in Histopathologic examination of target in minimum changes in the fluid of the a mixture, the mass and composition of organs in animals at all concentration lungs of the test animals. Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43827

(iii) At the appropriate time, the test (A) Chemical purity of the test (C) Results shall be quantified as animals shall be killed and the heart- material. amount of constituent/mL of lavage lung including trachea removed in bloc. (B) Airflow rates through the fluid. This assumes that the amount of Alternatively, lungs can be lavaged in inhalation equipment. lavage fluid recovered is a situ. If the study will not be (C) Temperature and humidity of air. representative sample of the total lavage compromised, one lobe of the lungs may (D) Nominal concentration (total fluid. be used for lung lavage while the other amount of test substance fed into the (3) Evaluation of data. The findings is fixed for histologic evaluation. The inhalation equipment divided by the from this acute study should be lungs should be lavaged using volume of air). evaluated in the context of preceding physiological saline. The lavages shall (E) Actual concentration in test and/or concurrent toxicity studies and consist of two washes, each of which breathing zone. any correlated functional findings. The consists of approximately 80% (e.g., 5 (F) Particle size distribution (e.g., evaluation shall include the relationship ml in rats and 1 ml in mice) of the total MMAD with GSD) and the bivariate between the concentrations of the test lung volume. Additional washes merely distribution of fiber length and substance and the presence or absence, tend to reduce the concentrations of the diameter, where appropriate. incidence, and severity of any effects. material collected. The lung lavage fluid (2) Results—(i) General group animal The evaluation should include shall be stored on ice at 5 °C until data. The following information shall be appropriate statistical analyses, for assayed. arranged by test group exposure level. example, parametric tests for (iv) The following parameters shall be (A) Number of animals exposed. continuous data and non-parametric determined in the lavage fluid as (B) Number of animals dying. tests for the remainder. Choice of indicators of cellular damage in the (C) Number of animals showing overt analyses should consider tests lungs: total protein, cell count, and signs of toxicity. appropriate to the experimental design, percent leukocytes. In addition, a (D) Pre- and post-exposure body including repeated measures. The report phagocytosis assay shall be performed weight change in animals, and weight must include concentration-response to determine macrophage activity. Assay change during the observation period. curves for the bronchoalveolar lavage (ii) Counts and incidence of gross methods described in the references and tables reporting observations at alterations observed at necropsy in the under paragraphs (h)(1) and (h)(3) of each concentration level for necropsy test and control groups. Data shall be this section may be used. findings and gross, general, and tabulated to show: (13) Combined protocol. The tests respiratory system histopathology. (A) The number of animals used in described may be combined with any each group and the number of animals (h) Reference. For additional other toxicity study, as long as none of in which any gross lesions were found. background information on this test the requirements of either are violated (B) The number of animals affected by guideline, the following references by the combination. each different type of lesion, and the should be consulted. These references (f) Triggered testing. If no adverse locations and frequency of each type of are available for inspection at the TSCA effects are seen in the 4-hr study as lesion. Nonconfidential Information Center, compared with controls, no further (iii) Counts and incidence of general Rm. NE–B607, Environmental testing is necessary. If the 4-hr study histologic alterations in the test group. Protection Agency, 401 M St., SW., shows positive effects in histopathology Data shall be tabulated to show: Washington, DC, 12 noon to 4 p.m., or the bronchoalveolar lavage, an 8-hr (A) The number of animals used in Monday through Friday, except legal study shall be conducted. Only those each group and the number of animals holidays. tissues showing positive results in the 4- in which any histopathologic lesions (1) Burleson, G.R., Fuller, L.B., hr study must be pursued in the follow- were found. Me¬nache, M.G., and Graham, J.A. Poly up 8-hr study. Similarly, if the option to (B) The number of animals affected by (I): poly (C)-enhanced alveolar perform a 1-hr study is exercised, only each different type of lesion, and the peritoneal macrophage phagocytosis: those tissues showing positive results in locations, frequency, and average grade Quantification by a new method the 4-hr study shall be pursued. of each type of lesion. utilizing fluorescent beads. Proceedings (g) Data reporting and evaluation. The (iv) Counts and incidence of of the Society of Experimental Biology final test report shall include the respiratory histopathologic alterations and Medicine. 184:468–476 (1987). following information: by the test group. Data shall be tabulated (2) Gardner, D.E., Crapo, J.D., and (1) Description of equipment and test to show: McClellan, R.O. (Eds.) Toxicology of the methods. A description of the general (A) The number of animals used in Lung. (Raven Press, New York, 1993) design of the experiment and any each group and the number of animals pp. i–xii, 1–30. equipment used shall be provided. in which any histopathologic lesions (3) Gilmour, G.I., and Selgrade, M.K. (i) Description of exposure apparatus, were found. A comparison of the pulmonary including design, type, dimensions, (B) The number of animals affected by defenses against streptococcal infection source of air, system for generating each different type of lesion, and the in rats and mice following O3 exposure: particles, aerosols, gasses, and vapors, locations, frequency, and average grade Differences in disease susceptibility and method of conditioning air, treatment of of each type of lesion. neutrophil recruitment. Toxicology and exhaust air, and the method of housing (v) Results of the bronchoalveolar Applied Pharmacology. 123:211–218 animals in a test chamber. lavage study. Data shall be tabulated to (1993). (ii) Description of the equipment for show: (4) Henderson, R.F., Benson, J.M., measuring temperature, humidity, and (A) The amount of administered Hahn, F.F., Hobbs, C.H., Jones, R.K., particulate aerosol concentration and lavage fluid and recovered lavage fluid Mauderly, J.L., McClellan, R.O., and size. for each test animal. Pickrell, J.A. New approaches for the (iii) Exposure data shall be tabulated (B) The magnitude of change of evaluation of pulmonary toxicity: and presented with mean values and biochemical and cytologic indices in Bronchoalveolar lavage fluid analysis. measure of variability (e.g., standard lavage fluids at each test concentration Fundamental and Applied Toxicology. deviation) and should include: for each animal. 5:451–458 (1985). 43828 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(5) Henderson, R.F. Use of chronic studies and establishing safety where the maximum attainable bronchoalveolar lavage to detect lung criteria for human exposure. Hazards of concentration produces no observable damage. Environmental Health inhaled substances are influenced by toxic effects. A full study using three Perspectives. 56:115–129 (1984). the inherent toxicity and by physical concentrations may not be necessary. (6) Henderson, R.F., Rebar, A.H., factors such as volatility and particle (e) Test procedures—(1) Animal Pickrell, J.A., and Newton, G.J. Early size. selection—(i) Species and strain. A damage indicators in the lung. III. (b) Source. The source material used mammalian species shall be used for Biochemical and cytological response of in developing this TSCA test guideline testing. A variety of rodent species may the lung to inhaled metal salts. is the OPPTS harmonized test guideline be used, although the rat is the preferred Toxicology and Applied Pharmacology. 870.3465 (June 1996 Public Draft). This species. Commonly used laboratory 50:123–136 (1979). source is available at the address in strains should be employed. If another (7) McClellan, R.O. and Henderson, paragraph (h) of this section. mammalian species is used, the tester R.F. (Eds.) Second edition. Concepts in (c) Definitions. The following shall provide justification/reasoning for Inhalation Toxicology. (Taylor and definitions apply to this section. its selection. Francis, Washington, DC, 1995) pp.i– Aerodynamic equivalent diameter is (ii) Age/weight. (A) Testing should be xxiv, 1–24, 441–470. defined as the diameter of a unit density started with young healthy animals as (8) Mery, S., Gross, E.A., Joyner, D.R., sphere having the same terminal settling soon as possible after weaning and Godo, M., and Morgan, K.T. Nasal velocity as the particle in question, acclimatization. Diagrams: A Tool for Recording the whatever its size, shape, and density. It (B) Exposure shall commence no later Distribution of Nasal Lesions in Rats is used to predict where in the than 8 weeks of age. and Mice. Toxicologic Pathology. respiratory tract such particles may be (C) At the commencement of the 22:353–372 (1994). deposited. study the weight variation of animals (9) Phalen, R.F. (Ed) Methods in Concentration in a subchronic used shall not exceed ± 20% of the mean Inhalation Toxicology. (CRC Press, Boca inhalation study is the amount of test weight for each sex. Raton, FL, 1997) pp. i–xii, 1–12. substance administered via inhalation (iii) Sex. (A) Equal numbers of (10) Renne, R.A., Gideon, K.M., for a period of 90–days. Concentration animals of each sex shall be used at Miller, R.A., Mellick, P.W., and is expressed as weight of the test each concentration. Grumbein, S.L. Histologic methods and substance per unit volume of air (B) Females shall be nulliparous and interspecies variations in the laryngeal (milligrams per liter or parts per nonpregnant. histology of F344/N rats and B6C3F1 million). (iv) Numbers. (A) At least 20 rodents mice. Toxicology and Pathology. 20:44– Cumulative toxicity is the adverse (10 females and 10 males) should be 51 (1992). effects of repeated exposures occurring used for each test group. If another (11) Young, J.T. Histopathologic as a result of prolonged action on, or mammalian species is selected (e.g. dog, examination of the rat nasal cavity. increased concentration of the rabbit, or nonhuman primate), at least Fundamental and Applied Toxicology. administered test substance or its eight animals per group (four males and 1:309–312 (1981). metabolites in susceptible tissues. Inhalable diameter refers to that four females) shall be used. § 799.9346 TSCA subchronic inhalation aerodynamic diameter of a particle (B) If interim sacrifices are planned, toxicity. which is considered to be inhalable for the number of animals shall be (a) Scope This section is intended to the organism. It is used to refer to increased by the number of animals meet the testing requirements under particles which are capable of being scheduled to be sacrificed before the section 4 of TSCA. In the assessment inhaled and may be deposited anywhere completion of the study. and evaluation of the toxic within the respiratory tract (C) To avoid bias, the use of adequate characteristics of a gas, volatile Mass median aerodynamic diameter randomization procedures for the substance, or aerosol/particulate, (MMAD) is the median aerodynamic proper allocation of animals to test and determination of subchronic inhalation diameter and along with the geometric control groups is required. toxicity may be carried out after initial standard deviation (GSD) is used to (D) Each animal shall be assigned a information on toxicity has been describe the particle size distribution of unique identification number. Dead obtained by acute testing. The any aerosol statistically based on the animals, their preserved organs and subchronic inhalation study has been weight and size of the particles. Fifty tissues, and microscopic slides shall be designed to permit the determination of percent of the particles by weight will identified by reference to the animal’s the no-observed-effect-level (NOEL) and be smaller than the median diameter unique number. toxic effects associated with continuous and 50% of the particles will be larger. (v) Husbandry. (A) Animals may be or repeated exposure to a test substance No-observed-effect-level (NOEL) is the group-caged by sex, but the number of for a period of 90 days. This study is not maximum concentration used in a study animals per cage must not interfere with capable of determining those effects that which produces no adverse effects. clear observation of each animal. The have a long latency period for Subchronic inhalation toxicity is the biological properties of the test development (e.g., carcinogenicity and adverse effects occurring as a result of substance or toxic effects (e.g., life shortening). Extrapolation from the the repeated daily exposure of morbidity, excitability) may indicate a results of this study to humans is valid experimental animals to a chemical by need for individual caging. Animals only to a limited degree. It can, inhalation for part (approximately 10%) must be housed individually in however, provide useful information on of a life span. inhalation chambers during exposure to health hazards likely to arise from (d) Limit test. The exposure is at a aerosols. repeated exposures by the inhalation concentration of 1 mg/L or greater (B) The temperature of the route over a limited period of time. It (expected human exposure may indicate experimental animal rooms should be at will provide information on target the need for a higher concentration), 22 ±3 °C. organs and the possibilities of where such concentration is not (C) The relative humidity of the accumulation, and can be of use in possible due to physical or chemical experimental animal rooms should be selecting concentration levels for properties of the test substance, or 30–70%. Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43829

(D) Where lighting is artificial, the vehicle at the highest exposure level) be maintained between 40 and 60%, but sequence should be 12 h light/12 h dark. shall be used. Concentrations should be in certain instances (e.g., use of water (E) Control and test animals should be spaced appropriately to produce test vehicle) this may not be practicable. fed from the same batch and lot. The groups with a range of toxic effects. The (9) Physical measurements. feed should be analyzed to assure data should be sufficient to produce a Measurements or monitoring shall be adequacy of nutritional requirements of concentration-response curve. made of the following: the species tested and for impurities (ii) The highest concentration should (i) The rate of airflow shall be that might influence the outcome of the result in toxic effects but not produce an monitored continuously but recorded at test. Animals should be fed and watered incidence of fatalities which would least three times during the exposure. ad libitum with food replaced at least prevent a meaningful evaluation. (ii) The actual concentrations of the weekly. For nonrodents feeding should (iii) The intermediate concentrations test substance shall be measured in the be at least daily and water ad libitum. should be spaced to produce a gradation animal’s breathing zone. During the (F) The study should not be initiated of toxic effects. exposure period, the actual until animals have been allowed a (iv) The lowest concentration should concentrations of the test substance period of acclimatization/quarantine. produce no evidence of toxicity. shall be held as constant as practicable (2) Control and test substances. (i) (v) In the case of potentially explosive and monitored continuously or Whenever it is necessary to formulate test substances, care should be taken to intermittently depending on the method the test substance with a vehicle for avoid generating explosive of analysis. Chamber concentration may aerosol generation, the vehicle ideally concentrations. be measured using gravimetric or should not elicit toxic effects or (6) Administration of the test analytical methods as appropriate. If substantially alter the chemical or substance. Animals should be exposed trial run measurements are reasonably toxicological properties of the test to the test substance for 6 h per day on consistent (± 10% for liquid, aerosol, substance. a 7–day per week basis for a period of gas, or vapor; ± 20% for dry aerosol), (ii) One lot of the test substance at least 90 days. Based primarily on then two measurements should be should be used, if possible throughout practical considerations, exposure for 6 sufficient. If measurements are not the duration of the study, and the h per day on a 5–day per week basis is consistent, three to four measurements research sample should be stored under acceptable. should be taken. Whenever the test conditions that maintain its purity and (7) Observation period. The animals substance is a formulation, or it is stability. Prior to the initiation of the should be observed for a period of 90 necessary to formulate the test study, there should be a characterization days. Animals in the satellite group (if substance with a vehicle for aerosol of the test substance, including the used) scheduled for follow-up generation, the analytical concentration purity of the test substance and, if observations should be kept for at least must be reported for the total technically feasible, the name and 28 days further without treatment to formulation, and not just for the active quantities of unknown contaminants assess reversibility. ingredient (AI). If, for example, a and impurities. (8) Exposure specifications. (i) The formulation contains 10% AI and 90% (3) Control groups. A concurrent animals shall be tested in dynamic inerts, a chamber analytical limit control group is required. This group inhalation equipment designed to concentration of 2 mg/L would consist shall be an untreated or sham-treated sustain a minimum airflow of 10 air of 0.2 mg/L of the AI. It is not necessary control group. Except for treatment with changes per hr, an adequate oxygen to analyze inert ingredients provided the test substance, animals in the content of at least 19%, and uniform the mixture at the animal’s breathing control group shall be handled in a conditions throughout the exposure zone is analogous to the formulation; manner identical to the test group chamber. Maintenance of slight negative the grounds for this conclusion must be animals. Where a vehicle other than pressure inside the chamber will provided in the study report. If there is water is used to generate a substance, a prevent leakage of the test substance some difficulty in measuring chamber vehicle control group should be used. If into the surrounding areas. It is not analytical concentration due to the toxic properties of the vehicle are normally necessary to measure chamber precipitation, nonhomogeneous not known or cannot be made available, oxygen concentration if airflow is mixtures, volatile components, or other both untreated and vehicle control adequate. factors, additional analyses of inert groups are required. (ii) The selection of a dynamic components may be necessary. (4) Satellite group. A satellite group of inhalation chamber should be (iii) During the development of the 20 animals (10 animals per sex) may be appropriate for the test substance and generating system, particle size analysis treated with the high concentration test system. Where a whole body shall be performed to establish the level for 90 days and observed for chamber is used to expose animals to an stability of aerosol concentrations with reversibility, persistence, or delayed aerosol, individual housing must be respect to particle size. The MMAD occurrence of toxic effects for a post- used to minimize crowding of the test particle size range should be between 1– treatment period of appropriate length, animals and maximize their exposure to 3 µm. The particle size of hygroscopic normally not less than 28 days. In the test substance. To ensure stability of materials should be small enough when addition, a control group of 20 animals a chamber atmosphere, the total volume dry to assure that the size of the swollen (10 animals of each sex) should be occupied by the test animals shall not particle will still be within the 1–3 µm added to the satellite study. exceed 5% of the volume of the test range. Measurements of aerodynamic (5) Concentration levels and chamber. It is recommended, but not particle size in the animal’s breathing concentration selection. (i) In required, that nose-only or head-only zone should be measured during a trial subchronic toxicity tests, it is desirable exposure be used for aerosol studies in run. If MMAD valves for each exposure to have a concentration-response order to minimize oral exposures due to level are within 10% of each other, then relationship as well as a NOEL. animals licking compound off their fur. two measurements during the exposures Therefore, at least three concentration Heat stress should be minimized. should be sufficient. If pretest levels plus a control and, where (iii) The temperature at which the test measurements are not within 10% of appropriate, a vehicle control is performed should be maintained at each other, three to four measurements (corresponding to the concentration of 22 ± 2 °C. The relative humidity should should be taken. 43830 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(iv) Temperature and humidity shall examined once prior to initiation of (ii) At least the liver, kidneys, brain, be monitored continuously and exposure, at monthly intervals or and gonads shall be trimmed and recorded at least three times during an midway through the test period and at weighed wet, as soon as possible after exposure. termination. dissection to avoid drying. (10) Feed and water during exposure (i) The recommended hematology (iii) The following organs and tissues, period. Feed shall be withheld during parameters are: Hemoglobin and or representative samples thereof, shall exposure. Water may also be withheld hematocrit concentrations, red blood be preserved in a suitable medium for during exposure. cell count, white blood cell count, possible future histopathological (11) Observation of animals. (i) differential leukocyte count, platelet examination: During and following exposure, count, and a measure of clotting (A) Digestive system. observations are made and recorded potential such as prothrombin time or (1) Salivary glands. systematically; individual records thromboplastin time. (2) Esophagus. should be maintained for each animal. (ii) Clinical chemistry parameters (3) Stomach. It is not always possible to observe which are considered appropriate to all (4) Duodenum. animals during exposure in a whole- studies are electrolyte balance, (5) Jejunum. body chamber. carbohydrate metabolism, and liver and (6) Ileum. (ii) Observations shall be made at kidney function. Other determinations (7) Cecum. least once each day for morbidity and which may be necessary for an adequate (8) Colon. mortality. Appropriate actions should toxicological evaluation include (9) Rectum. be taken to minimize loss of animals to analyses of lipids, hormones, acid/base (10) Liver. the study (e.g., Necropsy or refrigeration balance, methemoglobin and (11) Pancreas. of those animals found dead and cholinesterase activity. Additional (12) Gallbladder (dogs). isolation or sacrifice of weak or clinical biochemistry may be employed (B) Nervous system. moribund animals). where necessary to extend the (1) Brain (multiple sections). (iii) A careful clinical examination investigation of observed effects.The (2) Pituitary. shall be made at least once weekly. selection of specific tests will be (3) Peripheral nerve(s). Observations should be detailed and influenced by observations on the mode (4) Spinal cord (three levels). carefully recorded, preferably using of action of the substance and signs of (5) Eyes (retina, optic nerve). explicitly defined scales. Observations clinical toxicity. Suggested blood (C) Glandular system. should include, but not be limited to, clinical chemistry determinations: (1) Adrenals. evaluation of skin and fur, eyes and (A) Electrolytes. (2) Parathyroids. mucous membranes, respiratory and (1) Calcium. (3) Thyroids. circulatory effects, autonomic effects (2) Chloride. (D) Respiratory system. such as salivation, central nervous (3) Magnesium. (1) Trachea. system effects, including tremors and (4) Inorganic phosphorus. (2) Lung. convulsions, changes in the level of (5) Potassium. (3) Pharynx. activity, gait and posture, reactivity to (6) Sodium. (4) Larynx. handling or sensory stimuli, altered (B) Enzymes. (5) Nose. (1) Alkaline phosphatase. strength, and stereotypes or bizarre (E) Cardiovascular/hematopoietic (2) Alanine aminotransferase. system. behavior (e.g., self-mutilation, walking (3) Aspartate aminotransferase. backwards). (1) Aorta (thoracic). (4) Gamma glutamyl transferase. (2) Heart. (iv) Signs of toxicity should be (C) Other. (3) Bone marrow. recorded as they are observed including (1) Albumin. the time of onset, degree and duration. (2) Blood creatinine. (4) Lymph nodes. (v) Individual weights of animals (3) Blood urea nitrogen. (5) Spleen. shall be determined shortly before the (4) Globulins. (6) Thymus. test substance is administered, and (5) Glucose (fasting). (F) Urogenital system. weekly thereafter. (6) Total bilirubin. (1) Kidneys. (vi) Food consumption shall also be (7) Total cholesterol. (2) Urinary bladder. determined weekly if abnormal body (8) Total serum protein. (3) Prostate. weight changes are observed. (iii) Urinalysis is not recommended (4) Testes. (vii) Moribund animals should be on a routine basis, but only when there (5) Epididymides. removed and sacrificed when noticed is an indication based on expected or (6) Seminal vesicle(s). and the time of death should be observed toxicity. (7) Uterus. recorded as precisely as possible. (13) Ophthalmological examination. (8) Ovaries. (viii) At termination, all survivors in Ophthalmological examinations shall be (G) Other. the treatment groups shall be sacrificed. made on all animals prior to the (1) Lacrimal gland. (12) Clinical pathology. Hematology administration of the test substance and (2) Mammary gland. and clinical chemistry examinations on all high concentration and control (3) Skin. shall be made on all animals, including groups at termination. If changes in the (4) Skeletal muscle. controls, of each sex in each group for eyes are detected, all animals in the (5) All gross lesions and masses. rodents and all animals when other concentration groups shall be (6) Sternum and/or femur. nonrodents are used as test animals. For examined. (15) Histopathology. (i) The following rodents, the hematology and clinical (14) Gross pathology. (i) All animals histopathology shall be performed: chemistry parameters should be shall be subjected to a full gross (A) Full histopathology on the examined once prior to initiation of necropsy which includes examination respiratory tract and other organs and exposure and at terminal sacrifice. For of the external surface of the body, all tissues, listed under paragraph nonrodents, the hematology and clinical orifices and the cranial, thoracic, and (e)(15)(iii) of this section, of all animals chemistry parameters should be abdominal cavities and their contents. in the control and high exposure groups Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43831 and all animals that died or were killed study and provide information on the (A) Group animal data. Tabulation of during the study. selection of concentrations. toxic response data by species, strain, (B) All gross lesions in all animals. (3) Test report. In addition to sex and exposure level for: (C) Target organs in all animals. reporting requirements specified under (1) Number of animals exposed. (D) Lungs, liver and kidneys of all 40 CFR part 792, subpart J, the following (2) Number of animals showing signs animals. Special attention to specific information shall be reported. of toxicity. examination of the respiratory tract Both individual and summary data (3) Number of animals dying. should be made for evidence of should be presented. (B) Individual animal data. Data infection as this provides a convenient (i) Test substance characterization should be presented as summary (group assessment of the state of health of the shall include: mean) as well as for individual animals. animals. (A) Chemical identification. (1) Time of death during the study or (E) When a satellite group is used, (B) Lot or batch number. whether animals survived to histopathology shall be performed on (C) Physical properties. termination. (D) Purity/impurities. (2) Time of observation of each tissues and organs identified as showing (E) Identification and composition of effects in the treated groups. abnormal sign and its subsequent any vehicle used. course. (ii) If excessive early deaths or other (ii) Test system information shall problems occur in the high exposure (3) Body weight data. include: (4) Feed consumption data, when group compromising the significance of (A) Species and strain of animals used the data, the next concentration should collected. and rationale for selection if other than (5) Results of ophthalmological be examined for complete that recommended. examination, when performed. histopathology. (B) Age, sex, and body weight. (6) Results of hematological tests (iii) An attempt should be made to (C) Test environment including cage performed. . correlate gross observations with conditions, ambient temperature, (7) Results of clinical chemistry tests microscopic findings. humidity, and light/dark periods. performed. (iv) Tissues and organs designated for (iii) Test procedure information shall (8) Results of urinalysis tests microscopic examination should be include: performed. fixed in 10% buffered formalin or a (A) Method of randomization used. (9) Necropsy findings, including (B) Full description of experimental recognized suitable fixative as soon as absolute and relative organ weight data. necropsy is performed and no less than design and procedure. (10) Detailed description of all (C) Exposure regimen including 48 hrs prior to trimming. Tissues should histopathological findings. be trimmed to a maximum thickness of concentration levels, methods, and (11) Statistical treatment of results, 0.4 cm for processing. volume. where appropriate. (D) Description of test conditions; the (f) Data and reporting—(1) Treatment (g) Quality control. A system shall be following exposure conditions shall be of results. (i) Data shall be summarized developed and maintained to assure and reported: in tabular form, showing for each test document adequate performance of (1) Description of exposure apparatus group the number of animals at the start laboratory staff and equipment. The including design, type, volume, source of the test, the number of animals study shall be conducted in compliance of air, system for generating aerosols, showing lesions, the types of lesions, with 40 CFR Part 792—Good Laboratory method of conditioning air, treatment of and the percentage of animals Practice Standards. exhaust air and the method of housing displaying each type of lesion. (h) References. For additional (ii) All observed results (quantitative the animals in a test chamber. (2) The equipment for measuring background information on this test and qualitative) should be evaluated by guideline, the following references an appropriate statistical method. Any temperature, humidity, and particulate aerosol concentrations and size should should be consulted. These references generally accepted statistical method are available for inspection at the TSCA may be used; the statistical methods be described. (E) Exposure data shall be tabulated Nonconfidential Information Center, including significance criteria should be and presented with mean values and a Rm. NE–B607, Environmental selected during the design of the study. measure of variability (e.g., standard Protection Agency, 401 M St., SW., (2) Evaluation of study results. The deviation) and include: Washington, DC, 12 noon to 4 p.m., findings of the subchronic inhalation (1) Airflow rates through the Monday through Friday, except legal toxicity study should be evaluated in inhalation equipment. holidays. conjunction with the findings of (2) Temperature and humidity of air. (1) Cage, J.C. Ed. Paget, G.E. preceding studies and considered in (3) Actual (analytical or gravimetric) Experimental Inhalation Toxicology, terms of the observed toxic effects and concentration in the breathing zone. Methods in Toxicology. (F.A. Davis Co., the necropsy and histopathological (4) Nominal concentration (total Philadelphia, PA, 1970) pp. 258–277. findings. The evaluation will include amount of test substance fed into the (2) Casarett, L.J. and Doull. Chapter 9. the relationship between the inhalation equipment divided by Toxicology: The Basic Science of concentration of the test substance and volume of air). Poisons (New York: Macmillan duration of exposure, and the presence (5) Particle size distribution, Publishing Co., Inc., 1975). or absence, the incidence and severity, calculated mass median aerodynamic (3) U.S. Environmental Protection of abnormalities, including behavioral diameter (MMAD) and geometric Agency, Office of Pesticide Programs, and clinical abnormalities, gross lesions, standard deviation (GSD). Health Effects Division. Interim policy identified target organs, body weight (6) Explanation as to why the desired for particle size and limit concentration changes, effects on mortality and any chamber concentration and/or particle issues in inhalation toxicity studies other general or specific toxic effects. A size could not be achieved (if (February 1, 1994). properly conducted subchronic test applicable) and the efforts taken to (4) MacFarland, H.N. Ed. Hayes, W.J. should provide a satisfactory estimation comply with this aspect of the section. Vol. 7. Respiratory Toxicology, Essays in of a no-effect level. It also can indicate (iv) Test results information shall Toxicology. (Academic Press, New York, the need for an additional longer-term include: NY, 1976) pp. 121–154. 43832 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(5) Organisation for Economic Co- number of animals to yield characteristics: Effects on the operation and Development. Guidelines approximately 20 animals with absorption, distribution, metabolism, or for testing of chemicals, section 4-health implantation sites at necropsy. retention of the test substance; effects on effects, part 413. Subchronic Inhalation (2) Administration of test and control the chemical properties of the test Toxicity Studies (Paris, 1981). substances—(i) Dose levels and dose substance which may alter its toxic selection. (A) At least three-dose levels characteristics; and effects on the food § 799.9370 TSCA prenatal developmental and a concurrent control shall be used. or water consumption or the nutritional toxicity. Healthy animals shall be randomly status of the animals. (a) Scope This section is intended to assigned to the control and treatment (iii) Route of administration. (A) The meet the testing requirements under groups, in a manner which results in test substance or vehicle is usually section 4 of TSCA. This guideline for comparable mean body weight values administered orally by intubation. developmental toxicity testing is among all groups. The dose levels (B) If another route of administration designed to provide general information should be spaced to produce a gradation is used, for example, when the route of concerning the effects of exposure on of toxic effects. Unless limited by the administration is based upon the the pregnant test animal and on the physical/chemical nature or biological principal route of potential human developing organism; this may include properties of the test substance, the exposure, the tester shall provide death, structural abnormalities, or highest dose shall be chosen with the justification and reasoning for its altered growth and an assessment of aim to induce some developmental and/ selection, and appropriate modifications maternal effects. For information on or maternal toxicity but not death or may be necessary. Care should be taken testing for functional deficiencies and severe suffering. In the case of maternal to minimize stress on the maternal other postnatal effects, the guidelines mortality, this should not be more than animals. For materials administered by for the two-generation reproductive approximately 10%. The intermediate inhalation, whole-body exposure is toxicity study and the developmental dose levels should produce minimal preferable to nose-only exposure due to neurotoxicity study should be observable toxic effects. The lowest dose the stress of restraint required for nose- consulted. level should not produce any evidence only exposure. (b) Source. The source material used of either maternal or developmental (C) The test substance shall be in developing this TSCA test guideline toxicity (i.e., the no-observed-adverse- administered at approximately the same is the OPPTS harmonized test guideline effect level, NOAEL) or should be at or time each day. 870.3700 (February 1996 Public Draft). near the limit of detection for the most (D) When administered by gavage or This source is available at the address sensitive endpoint. Two- or four-fold dermal application, the dose to each in paragraph (h) of this section. intervals are frequently optimal for animal shall be based on the most recent (c) Good laboratory practice spacing the dose levels, and the individual body weight determination. standards. The study shall be conducted addition of a fourth test group is often (iv) Dosing schedule. At minimum, in compliance with 40 CFR Part 792— preferable to using very large intervals the test substance shall be administered Good Laboratory Practice Standards. (e.g., more than a factor of 10) between daily from implantation to the day (d) Principle of the test method. The dosages. test substance is administered to (B) It is desirable that additional before cesarean section on the day prior pregnant animals at least from information on metabolism and to the expected day of parturition. implantation to one day prior to the pharmacokinetics of the test substance Alternatively, if preliminary studies do expected day of parturition. Shortly be available to demonstrate the not indicate a high potential for before the expected date of delivery, the adequacy of the dosing regimen. This preimplantation loss, treatment may be pregnant females are terminated, the information should be available prior to extended to include the entire period of uterine contents are examined, and the testing. gestation, from fertilization to fetuses are processed for visceral and (C) The highest dose tested need not approximately 1 day prior to the skeletal evaluation. exceed 1,000 mg/kg/day by oral or expected day of termination. (e) Test procedures—(1) Animal dermal administration, or 2 mg/L (or the (f) Observation of animals—(1) selection—(i) Species and strain. It is maximum attainable concentration) by Maternal. (i) Each animal shall be recommended that testing be performed inhalation, unless potential human observed at least once daily, considering in the most relevant species, and that exposure data indicate the need for the peak period of anticipated effects laboratory species and strains which are higher doses. If a test performed at the after dosing. Mortality, moribundity, commonly used in prenatal limit dose level, using the procedures pertinent behavioral changes, and all developmental toxicity testing be described for this study, produces no signs of overt toxicity shall be recorded employed. The preferred rodent species observable toxicity and if an effect at this cageside observation. In addition, is the rat and the preferred non-rodent would not be expected based upon data thorough physical examinations shall be species is the rabbit. from structurally related compounds, conducted at the same time maternal (ii) Age. Young adult animals shall be then a full study using three-dose levels body weights are recorded. used. may not be considered necessary. (ii) Animals shall be weighed on day (iii) Sex. Nulliparous female animals (ii) Control group. (A) A concurrent 0, at termination, and at least at 3–day shall be used at each dose level. control group shall be used. This group intervals during the dosing period. Animals should be mated with males of shall be a sham-treated control group or (iii) Food consumption shall be the same species and strain, avoiding a vehicle-control group if a vehicle is recorded on at least 3-day intervals, the mating of siblings, if parentage is used in administering the test preferably on days when body weights known. Day 0 in the test is the day on substance. are recorded. which a vaginal plug and/or sperm are (B) The vehicle control group should (iv) (A) Females shall be terminated observed in the rodent or that receive the vehicle in the highest immediately prior to the expected day insemination is performed or observed volume used. of delivery. in the rabbit. (C) If a vehicle or other additive is (B) Females showing signs of abortion (iv) Number of animals. Each test and used to facilitate dosing, consideration or premature delivery prior to control group shall contain a sufficient should be given to the following scheduled termination shall be killed Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43833 and subjected to a thorough form, showing for each test group the (A) Corpora lutea counts. macroscopic examination. types of change and the number of (B) Implantation data, number and (v) At the time of termination or death dams, fetuses, and litters displaying percent of live and dead fetuses, and during the study, the dam shall be each type of change. resorptions (early and late). examined macroscopically for any (2) Evaluation of study results. The (C) Pre- and postimplantation loss structural abnormalities or pathological following shall be provided: calculations. changes which may have influenced the (i) Maternal and fetal test results, (iv) Developmental endpoints by dose pregnancy. Evaluation of the dams including an evaluation of the for litters with live fetuses, including: during cesarean section and subsequent relationship, or lack thereof, between (A) Number and percent of live fetal analyses should be conducted the exposure of the animals to the test offspring. without knowledge of treatment group substance and the incidence and (B) Sex ratio. in order to minimize bias. severity of all findings. (C) Fetal body weight data, preferably (vi) (A) Immediately after termination (ii) Criteria used for categorizing fetal by sex and with sexes combined. or as soon as possible after death, the external, soft tissue, and skeletal (D) External, soft tissue, and skeletal uteri shall be removed and the anomalies. malformation and variation data. The pregnancy status of the animals (iii) When appropriate, historical total number and percent of fetuses and ascertained. Uteri that appear nongravid control data to enhance interpretation of litters with any external, soft tissue, or shall be further examined (e.g. by study results. Historical data (on litter skeletal alteration, as well as the types ammonium sulfide staining) to confirm incidence and fetal incidence within and incidences of individual anomalies, the nonpregnant status. litter), when used, should be compiled, should be reported. (B) Each gravid uterus (with cervix) presented, and analyzed in an (v) The numbers used in calculating shall be weighed. Gravid uterine appropriate and relevant manner. In all percentages or indices. weights should not be obtained from order to justify its use as an analytical (vi) Adequate statistical treatment of dead animals if autolysis or tool, information such as the dates of results. decomposition has occurred. study conduct, the strain and source of (vii) A copy of the study protocol and (C) The number of corpora lutea shall the animals, and the vehicle and route any amendments should be included. be determined for pregnant animals. of administration should be included. (h) References. For additional (D) The uterine contents shall be (iv) Statistical analysis of the study background information on this test examined for embryonic or fetal deaths findings should include sufficient guideline, the following references and the number of viable fetuses. The information on the method of analysis, should be consulted. These references degree of resorption shall be described so that an independent reviewer/ are available for inspection at the TSCA in order to help estimate the relative statistician can reevaluate and Nonconfidential Information Center, time of death of the conceptus. reconstruct the analysis. In the Rm. NE–B607, Environmental (2) Fetal. (i) The sex and body weight evaluation of study data, the litter Protection Agency, 401 M St., SW., of each fetus shall be determined. should be considered the basic unit of Washington, DC, 12 noon to 4 p.m., (ii) Each fetus shall be examined for analysis. Monday through Friday, except legal external anomalies. (v) In any study which demonstrates holidays. (iii) Fetuses shall be examined for an absence of toxic effects, further (1) Aliverti, V.L. et al. The extent of skeletal and soft tissue anomalies (e.g. investigation to establish absorption and fetal ossification as an index of delayed variations and malformations or other bioavailability of the test substance development in teratogenicity studies in categories of anomalies as defined by should be considered. the rat. Teratology. 20:237–242 (1979). the performing laboratory). (3) Test report. In addition to the (2) Barrow, M.V. and W.J. Taylor. A (A) For rodents, approximately one- reporting requirements as specified rapid method for detecting half of each litter shall be prepared by under 40 CFR part 792, subpart J, the malformations in rat fetuses. Journal of standard techniques and examined for following specific information shall be Morphology 127:291–306 (1969). skeletal alterations, preferably bone and reported. Both individual and summary (3) Burdi, A.R. Toluidine blue-alizarin cartilage. The remainder shall be data should be presented. red S staining of cartilage and bone in prepared and examined for soft tissue (i) Species and strain. whole-mount skeltons in vitro. Stain (ii) Maternal toxic response data by anomalies, using appropriate serial Technolology. 40:45–48 (1965). dose, including but not limited to: sectioning or gross dissection (A) The number of animals at the start (4) Edwards, J.A. Ed. Woolam,D.H.M. techniques. It is also acceptable to of the test, the number of animals The external development of the rabbit examine all fetuses by careful dissection surviving, the number pregnant, and the and rat embryo. Vol. 3. Advances in for soft tissue anomalies followed by an number aborting. Teratology (Academic, NY, 1968). examination for skeletal anomalies. (B) Day of death during the study or (5) Fritz, H. Prenatal ossification in (B) For rabbits, all fetuses shall be whether animals survived to rabbits as indicative of fetal maturity. examined for both soft tissue and termination. Teratology. 11:313–320 (1974). skeletal alterations. The bodies of these (C) Day of observation of each (6) Fritz, H. and Hess, R. Ossification fetuses should be evaluated by careful abnormal clinical sign and its of the rat and mouse skeleton in the dissection for soft-tissue anomalies, subsequent course. perinatal period. Teratology. 3:331–338 followed by preparation and (D) Body weight and body weight (1970). examination for skeletal anomalies. An change data, including body weight (7) Gibson, J.P. et al. Use of the rabbit adequate evaluation of the internal change adjusted for gravid uterine in teratogenicity studies. Toxicology structures of the head, including the weight. and Applied Pharmacology. 9:398–408 eyes, brain, nasal passages, and tongue, (E) Food consumption and, if (1966). should be conducted for at least half of applicable, water consumption data. (8) Inouye, M. Differential staining of the fetuses. (F) Necropsy findings, including cartilage and bone in fetal mouse (g) Data and reporting—(1) Treatment gravid uterine weight. skeleton by alcian blue and alizarin red of results. Data shall be reported (iii) Developmental endpoints by dose S. Congenital Anomalies. 16(3):171–173 individually and summarized in tabular for litters with implants, including: (1976). 43834 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(9) Igarashi, E. et al. Frequence of fetal bone. Stain Technology. 39:61–63 (d) Principle of the test method. The spontaneous axial skeletal variations (1964). test substance is administered to detected by the double staining (23) Strong, R.M. The order time and parental (P) animals prior to and during technique for ossified and cartilaginous rate of ossification of the albino rat (mus their mating, during the resultant skeleton in rat fetuses. Congenital norvegicus albinus) skeleton. American pregnancies, and through the weaning Anomalies. 32:381–391 (1992). Journal of Anatomy. 36: 313–355 (1928). of their F1 offspring. The substance is (10) Kimmel, C.A. et al. Skeletal (24) Stuckhardt, J.L. and Poppe, S.M. then administered to selected F1 development following heat exposure in Fresh visceral examination of rat and offspring during their growth into the rat. Teratology. 47:229–242 (1993). rabbit fetuses used in teratogenicity adulthood, mating, and production of an (11) Kimmel, C.A. and Francis, E.Z. testing. Teratogenesis, Carcinogenesis, F2 generation, until the F2 generation is Proceedings of the workshop on the and Mutagenesis. 4:181–188 (1984). weaned. acceptability and interpretation of (25) Van Julsingha, E.B. and (e) Test procedures—(1) Animal dermal developmental toxicity studies. Bennett,C.G. Eds. Neubert, D., Merker, selection—(i) Species and strain. The rat Fundamental and Applied Toxicology. H.J., and Kwasigroch, T.E. A dissecting is the most commonly used species for 14:386–398 (1990). procedure for the detection of anomalies testing. If another mammalian species is (12) Kimmel, C.A. and C. Trammell. A in the rabbit foetal head. Methods in used, the tester shall provide rapid procedure for routine double Prenatal Toxicology (University of justification/reasoning for its selection, staining of cartilage and bone in fetal Chicago, Chicago, IL, 1977) pp. 126–144 and appropriate modifications will be and adult animals. Stain Technology. . necessary. Healthy parental animals, which have been acclimated to 56:271–273 (1981). (26) Whitaker, J. and Dix, D.M. laboratory conditions for at least 5 days (13) Kimmel, C.A. and Wilson, J.G. Double-staining for rat foetus skeletons and have not been subjected to previous Skeletal deviation in rats: malformations in teratological studies. Laboratory or variations? Teratology. 8:309–316 experimental procedures, should be Animals. 13:309–310 (1979). used. Strains of low fecundity shall not (1973). (27) Wilson, J.G. Eds. Wilson, J.G. and (14) Marr, M.C. et al. Comparison of be used. Warkany, J. Embryological (ii) Age. Parental (P) animals shall be single and double staining for considerations in teratology. Teratology: 5 to 9 weeks old at the start of dosing. evaluation of skeletal development: the Principles and Techniques (University The animals of all test groups should be effects of ethylene glycol (EG) in CD of Chicago, Chicago, IL, 1965) pp. 251– of uniform weight, age, and parity as rats. Teratology. 37:476 (1988). 277. nearly as practicable, and should be (15) Marr, M.C. et al. Developmental representative of the species and strain stages of the CD (Sprague-Dawley) rat § 799.9380 TSCA reproduction and fertility effects. under study. skeleton after maternal exposure to (iii) Sex. (A) For an adequate ethylene glycol. Teratology. 46:169–181 (a) Scope. This section is intended to assessment of fertility, both males and (1992). meet the testing requirements under females shall be studied. (16) McLeod, M.J. Differential staining section 4 of the TSCA. This section is (B) The females shall be nulliparous of cartilage and bone in whole mouse for two-generation reproduction testing and nonpregnant. fetuses by Alcian blue and alizarin red and is designed to provide general (iv) Number of animals. Each control S. Teratology. 22:299–301 (1980). information concerning the effects of a group shall contain a sufficient number (17) Monie, I.W. et al. Dissection test substance on the integrity and of mating pairs to yield approximately procedures for rat fetuses permitting performance of the male and female 20 pregnant females. Each test group alizarin red staining of skeleton and reproductive systems, including gonadal shall contain a similar number of mating histological study of viscera. function, the estrous cycle, mating pairs. Supplement to Teratology Workshop behavior, conception, gestation, (v) Identification of animals. Each Manual. pp. 163–173 (1965). parturition, lactation, and weaning, and animal shall be assigned a unique (18) Organisation for Economic Co- on the growth and development of the identification number. For the P operation and Development, No. 414: offspring. The study may also provide generation, this should be done before Teratogenicity, Guideline for Testing of information about the effects of the test dosing starts. For the F1 generation, this Chemicals. [C(83)44 (Final)] (1983). substance on neonatal morbidity, should be done for animals selected for (19) Salewski (Koeln), V.E. mortality, target organs in the offspring, mating; in addition, records indicating Faerbermethode zum makroskopischen and preliminary data on prenatal and the litter of origin shall be maintained nachweis von implantations stellen am postnatal developmental toxicity and for all selected F1 animals. uterus der ratte. Naunyn-Schmeidebergs serve as a guide for subsequent tests. (2) Administration of test and control Archiv fu¨ r Pharmakologie und Additionally, since the study design substances—(i) Dose levels and dose Experimentelle Pathologie. 247:367 includes in utero as well as postnatal selection. (A) At least three-dose levels (1964). exposure, this study provides the and a concurrent control shall be used. (20) Spark, C. and Dawson,A.B. The opportunity to examine the Healthy animals should be randomly order and time of appearance of centers susceptibility of the immature/neonatal assigned to the control and treatment of ossification in the fore and hind animal. groups, in a manner which results in limbs of the albino rat, with special (b) Source. The source material used comparable mean body weight values reference to the possible influence of the in developing this TSCA test guideline among all groups. The dose levels sex factor. American Journal of is the OPPTS harmonized test guideline should be spaced to produce a gradation Anatomy. 41:411–445 (1928). 870.3800 (February 1996 Public Draft). of toxic effects. Unless limited by the (21) Staples, R.E. Detection of visceral This source is available at the address physical/chemical nature or biological alterations in mammalian fetuses. in paragraph (g) of this section. properties of the test substance, the Teratology. 9(3):A37–A38 (1974). (c) Good laboratory practice highest dose should be chosen with the (22) Staples, R.E. and Schnell, V.L. standards. The study shall be conducted aim to induce some reproductive and/or Refinements in rapid clearing technique in compliance with 40 CFR Part 792— systemic toxicity but not death or severe in the KOH—alizarin red S method for Good Laboratory Practice Standards. suffering. In the case of parental Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43835 mortality, this should not be more than lactation shall be based on the weeks following weaning of the last F1a approximately 10%. The intermediate individual animal body weight and or F2a litter. dose levels should produce minimal adjusted weekly at a minimum. (iv) Special housing. After evidence of observable toxic effects. The lowest dose (C) If another route of administration copulation, animals that are presumed level should not produce any evidence is used, for example, when the route of to be pregnant shall be caged separately of either systemic or reproductive administration is based upon the in delivery or maternity cages. Pregnant toxicity (i.e., the no-observed-adverse- principal route of potential human animals shall be provided with nesting effect level, NOAEL) or should be at or exposure, the tester should provide materials when parturition is near. near the limit of detection for the most justification and reasoning for its (v) Standardization of litter sizes. (A) sensitive endpoint. Two- or four-fold selection, and appropriate modifications Animals should be allowed to litter intervals are frequently optimal for may be necessary. Care should be taken normally and rear their offspring to spacing the dose levels, and the to minimize stress on the maternal weaning. Standardization of litter sizes addition of a fourth test group is often animals and their litters during is optional. preferable to using very large intervals gestation and lactation. (B) If standardization is performed, (e.g., more than a factor of 10) between (D) All animals should be dosed by the following procedure should be used. dosages. the same method during the appropriate On day 4 after birth, the size of each (B) It is desirable that additional experimental period. litter may be adjusted by eliminating information on metabolism and (iv) Dosing schedule. (A) The animals extra pups by random selection to yield, pharmacokinetics of the test substance should be dosed with the test substance as nearly as possible, four males and be available to demonstrate the on a 7–days–a–week basis. four females per litter or five males and adequacy of the dosing regimen. This (B) Daily dosing of the parental (P) five females per litter. Selective information should be available prior to males and females shall begin when elimination of pups, i.e. based upon testing. they are 5 to 9 weeks old. Daily dosing body weight, is not appropriate. (C) The highest dose tested should not of the F1 males and females shall begin Whenever the number of male or female exceed 1,000 mg/kg/day (or 20,000 ppm at weaning. For both sexes (P and F1), pups prevents having four (or five) of in the diet), unless potential human dosing shall be continued for at least 10 each sex per litter, partial adjustment exposure data indicate the need for weeks before the mating period. (for example, five males and three higher doses. If a test performed at the (C) Daily dosing of the P and F1 males females, or four males and six females) limit dose level, using the procedures and females shall continue until is acceptable. Adjustments are not described for this study, produces no termination. appropriate for litters of eight pups or observable toxicity and if an effect (3) Mating procedure—(i) Parental. less. would not be expected based upon data (A) For each mating, each female shall (4) Observation of animals—(i) from structurally related compounds, be placed with a single randomly Parental. (A) Throughout the test then a full study using three dose levels selected male from the same dose level period, each animal shall be observed at may not be considered necessary. (1:1 mating) until evidence of least once daily, considering the peak (ii) Control group. (A) A concurrent copulation is observed or either 3 period of anticipated effects after control group shall be used. This group estrous periods or 2 weeks has elapsed. dosing. Mortality, moribundity, shall be an untreated or sham treated Animals should be separated as soon as pertinent behavioral changes, signs of group or a vehicle-control group if a possible after evidence of copulation is difficult or prolonged parturition, and vehicle is used in administering the test observed. If mating has not occurred all signs of overt toxicity shall be substance. after 2 weeks or 3 estrous periods, the recorded at this cageside examination. (B) If a vehicle is used in animals should be separated without In addition, thorough physical administering the test substance, the further opportunity for mating. Mating examinations should be conducted control group shall receive the vehicle pairs should be clearly identified in the weekly on each animal. in the highest volume used. data. (B) Parental animals (P and F1) shall (C) If a vehicle or other additive is (B) Vaginal smears shall be collected be weighed on the first day of dosing used to facilitate dosing, consideration daily and examined for all females and weekly thereafter. Parental females should be given to the following during mating, until evidence of (P and F1) should be weighed at a characteristics: Effects on the copulation is observed. minimum on approximately gestation absorption, distribution, metabolism, or (C) Each day, the females shall be days 0, 7, 14, and 21, and during retention of the test substance; effects on examined for presence of sperm or lactation on the same days as the the chemical properties of the test vaginal plugs. Day 0 of pregnancy is weighing of litters. substance which may alter its toxic defined as the day a vaginal plug or (C) During the premating and characteristics; and effects on the food sperm are found. gestation periods, food consumption or water consumption or the nutritional (ii) F1 mating. For mating the F1 shall be measured weekly at a status of the animals. offspring, at least one male and one minimum. Water consumption should (D) If a test substance is administered female should be randomly selected be measured weekly at a minimum if in the diet and causes reduced dietary from each litter for mating with another the test substance is administered in the intake or utilization, the use of a pair- pup of the same dose level but different water. fed control group may be considered litter, to produce the F2 generation. (D) Estrous cycle length and normality necessary. (iii) Second mating. In certain should be evaluated by vaginal smears (iii) Route of administration. (A) The instances, such as poor reproductive for all P and F1 females during a test substance is usually administered performance in the controls, or in the minimum of 3 weeks prior to mating by the oral route (diet, drinking water, event of treatment-related alterations in and throughout cohabitation; care or gavage). litter size, the adults may be remated to should be taken to prevent the (B) If administered by gavage or produce an F1b or F2b litter. If induction of pseudopregnancy. dermal application, the dosage production of a second litter is deemed (E) For all P and F1 males at administered to each animal prior to necessary in either generation, the dams termination, sperm from one testis and mating and during gestation and should be remated approximately 1–2 one epididymis shall be collected for 43836 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations enumeration of homogenization- isolated heads, and misshapen heads (with coagulating glands and their resistant spermatids and cauda and/or tails. Evaluation of only control fluids), and prostate. epididymal sperm reserves, and high-dose P and F1 males may be (C) Brain, pituitary, liver, kidneys, respectively. In addition, sperm from performed unless treatment-related adrenal glands, spleen, and known the cauda epididymis (or vas deferens) effects are observed; in that case, the target organs. should be collected for evaluation of lower dose groups should also be (ii) For F1 and F2 weanlings that are sperm motility and sperm morphology. evaluated. examined macroscopically, the (1) The total number of (ii) Offspring. (A) Each litter should be following organs shall be weighed for homogenization-resistant testicular examined as soon as possible after one randomly selected pup per sex per sperm and cauda epididymal sperm delivery (lactation day 0) to establish litter. should be enumerated. The method the number and sex of pups, stillbirths, (A) Brain. described in the reference under live births, and the presence of gross (B) Spleen and thymus. paragraph (g)(8) of this section may be anomalies. Pups found dead on day 0 (8) Tissue preservation. The following used. Cauda sperm reserves can be should be examined for possible defects organs and tissues, or representative derived from the concentration and and cause of death. samples thereof, shall be fixed and volume of sperm in the suspension used (B) Live pups should be counted, stored in a suitable medium for to complete the qualitative evaluations, sexed, and weighed individually at histopathological examination. and the number of sperm recovered by birth, or soon thereafter, at least on days (i) For the parental (P and F1) subsequent mincing and/or 4, 7, 14, and 21 of lactation, at the time animals: homogenizing of the remaining cauda of vaginal patency or balanopreputial (A) Vagina, uterus with oviducts, tissue. Enumeration in only control and separation, and at termination. cervix, and ovaries. high-dose P and F1 males may be (C) The age of vaginal opening and (B) One testis (preserved in Bouins performed unless treatment-related preputial separation should be fixative or comparable preservative), effects are observed; in that case, the determined for F1 weanlings selected one epididymis, seminal vesicles, lower dose groups should also be for mating. If there is a treatment-related prostate, and coagulating gland. evaluated. effect in F1 sex ratio or sexual (C) Pituitary and adrenal glands. (2) An evaluation of epididymal (or maturation, anogenital distance should (D) Target organs, when previously vas deferens) sperm motility should be be measured on day 0 for all F2 pups. identified, from all P and F1 animals performed. Sperm should be recovered (5) Termination schedule. (i) All P selected for mating. while minimizing damage (the and F1 adult males and females should (E) Grossly abnormal tissue. evaluation techniques as described in be terminated when they are no longer (ii) For F1 and F2 weanlings selected the reference under paragraph (g)(8) of needed for assessment of reproductive for macroscopic examination: Grossly this section may be used), and the effects. abnormal tissue and target organs, when percentage of progressively motile (ii) F1 offspring not selected for known. sperm should be determined either mating and all F2 offspring should be (9) Histopathology—(i) Parental subjectively or objectively. For objective terminated at comparable ages after animals. Full histopathology of the evaluations, an acceptable counting weaning. organs listed under paragraph (e)(8)(i) of chamber of sufficient depth can be used (6) Gross necropsy. (i) At the time of this section shall be performed for ten to effectively combine the assessment of termination or death during the study, randomly chosen high dose and control motility with sperm count and sperm all parental animals (P and F1) and P and F1 animals per sex, for those morphology. When computer-assisted when litter size permits at least three animals that were selected for mating. motion analysis is performed, the pups per sex per litter from the Organs demonstrating treatment-related derivation of progressive motility relies unselected F1 weanlings and the F2 changes shall also be examined for the on user-defined thresholds for average weanlings shall be examined remainder of the high-dose and control path velocity and straightness or linear macroscopically for any structural animals and for all parental animals in index. If samples are videotaped, or abnormalities or pathological changes. the low- and mid-dose groups. images otherwise recorded, at the time Special attention shall be paid to the Additionally, reproductive organs of the of necropsy, subsequent analysis of only organs of the reproductive system. low- and mid-dose animals suspected of control and high-dose P and F1 males (ii) Dead pups or pups that are reduced fertility, e.g., those that failed to may be performed unless treatment- terminated in a moribund condition mate, conceive, sire, or deliver healthy related effects are observed; in that case, should be examined for possible defects offspring, or for which estrous cyclicity the lower dose groups should also be and/or cause of death. or sperm number, motility, or evaluated. In the absence of a video or (iii) At the time of necropsy, a vaginal morphology were affected, shall be digital image, all samples in all smear should be examined to determine subjected to histopathological treatment groups should be analyzed at the stage of the estrous cycle. The uteri evaluation. Besides gross lesions such as necropsy. of all cohabited females should be atrophy or tumors, testicular (3) A morphological evaluation of an examined, in a manner which does not histopathological examination should epididymal (or vas deferens) sperm compromise histopathological be conducted in order to to identify sample shall be performed. Sperm (at evaluation, for the presence and number treatment-related effects such as least 200 per sample) should be of implantation sites. retained spermatids, missing germ cell examined as fixed, wet preparations (the (7) Organ weights. (i) At the time of layers or types, multinucleated giant techniques for such examinations is termination, the following organs of all cells, or sloughing of spermatogenic described in the references under P and F1 parental animals shall be cells into the lumen. Examination of the paragraphs (g)(4) and (g)(8) of this weighed: intact epididymis should include the section may be used) and classified as (A) Uterus (with oviducts and cervix), caput, corpus, and cauda, which can be either normal (both head and midpiece/ ovaries. accomplished by evaluation of a tail appear normal) or abnormal. (B) Testes, epididymides (total longitudinal section, and should be Examples of morphologic sperm weights for both and cauda weight for conducted in order to identify such abnormalities would include fusion, either one or both), seminal vesicles lesions as sperm granulomas, leukocytic Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43837 infiltration (inflammation), aberrant cell the animals, and the vehicle and route (xiii) Implantation data and types within the lumen, or the absence of administration should be included. postimplantation loss calculations for P of clear cells in the cauda epididymal (iii) Statistical analysis of the study and F1 parental females. epithelium. The postlactational ovary findings should include sufficient (xiv) Absolute and adjusted organ should contain primordial and growing information on the method of analysis, weight data. follicles as well as the large corpora so that an independent reviewer/ (xv) Detailed description of all lutea of lactation. Histopathological statistician can reevaluate and histopathological findings. examination should detect qualitative reconstruct the analysis. (xvi) Adequate statistical treatment of depletion of the primordial follicle (iv) In any study which demonstrates results. population. A quantitative evaluation of an absence of toxic effects, further (xvii) A copy of the study protocol primordial follicles should be investigation to establish absorption and and any amendments should be conducted for all F1 females if any of bioavailability of the test substance included. the following treatment-related findings should be considered. (g) References. For additional were observed: (3) Test report. In addition to the backgound information on this test (A) Reductions in ovarian weight and reporting requirements as specified guideline, the following references abnormal ovarian histopathology under 40 CFR part 792, subpart J, the should be consulted. These references findings, e.g., follicular cysts or following specific information shall be are available for inspection at the TSCA qualitative evidence of a reduced reported. Both individual and summary Nonconfidential Information Center, population of primordial follicles. data should be presented. Rm. NE–B607, Environmental (B) Abnormal estrous cyclicity and (i) Species and strain. Protection Agency, 401 M St., SW., female infertility. (ii) Toxic response data by sex and Washington, DC, 12 noon to 4 p.m., (C) Depletion of testicular spermatid dose, including indices of mating, Monday through Friday, except legal counts in F1 males and evidence of fertility, gestation, birth, viability, and holidays. (1) Gray, L.E. et al. A dose-response germ cell depletion in testicular lactation; offspring sex ratio; precoital analysis of methoxychlor-induced histopathology evaluations. interval, including the number of days alterations of reproductive development (ii) Examination of ovarian sections. If until mating and the number of estrous and function in the rat. Fundamental a quantitative evaluation is performed, periods until mating; and duration of and Applied Toxicology. 12:92–108 ten ovarian sections shall be taken at gestation calculated from day 0 of (1989). µ pregnancy. The report should provide least 100 m apart from the inner third (2) Heindel, J.J. et al. Ed. Hirshfield, the numbers used in calculating all of each ovary. Examination should A.N. Histological assessment of ovarian indices. include enumeration of the total number follicle number in mice as a screen of of primordial and antral follicles from (iii) Day (week) of death during the ovarian toxicity. Growth Factors and the these 20 sections (the technique for this study or whether animals survived to Ovary (Plenum, NY, 1989) pp. 421–426. histological assessment as described in termination; date (age) of litter (3) Korenbrot, C.C. et al. Preputial the reference under paragraph (g)(2) of termination. separation as an external sign of this section may be used) for (iv) Toxic or other effects on pubertal development in the male rat. comparison with control ovaries. reproduction, offspring, or postnatal Biology of Reproduction. 17:298–303 (iii) Weanlings. For F1 and F2 growth. (1977). weanlings, histopathological (v) Developmental milestone data (4) Linder, R.E. et al. Endpoints of examination of treatment-related (mean age of vaginal opening and spermatoxicity in the rat after short abnormalities noted at macroscopic preputial separation, and mean duration exposures to fourteen examination should be considered, if anogenital distance, when measured). reproductive toxicants. Reproductive such evaluation were deemed (vi) Number of P and F1 females Toxicology. 6:491–505 (1992). appropriate and would contribute to the cycling normally and mean estrous (5) Manson, J.M. and Kang, Y.J. Ed. interpretation of the study data. cycle length. Hayes, A.W. Test methods for assessing (f) Data and reporting—(1) Treatment (vii) Day (week) of observation of each female reproductive and developmental of results. Data shall be reported abnormal sign and its subsequent toxicology. Principles and Methods of individually and summarized in tabular course. Toxicology (Raven, NY, 1989). form, showing for each test group the (viii) Body weight and body weight (6) Organisation for Economic Co- types of change and the number of change data by sex for P, F1, and F2 operation and Development, No. 416: animals displaying each type of change. animals. Two Generation Reproduction Toxicity (2) Evaluation of study results. (i) An (ix) Food (and water, if applicable) Study, Guidelines for Testing of evaluation of test results, including the consumption, food efficiency (body Chemicals. [C(83)44 (Final)] (1983). statistical analysis, shall be provided. weight gain per gram of food (7) Pederson, T. and Peters, H. This should include an evaluation of the consumed), and test material Proposal for classification of oocytes relationship, or lack thereof, between consumption for P and F1 animals, and follicles in the mouse ovary. Journal the exposure of the animals to the test except for the period of cohabitation. of Reproduction and Fertility. 17:555– substance and the incidence and (x) Total cauda epididymal sperm 557 (1988). severity of all abnormalities. number, homogenization-resistant testis (8) Seed, J., Chapin, R.E. E.D. Clegg, (ii) When appropriate, historical spermatid number, number and percent L.A. Dostal, R.H. Foote, M.E. Hurtt, G.R. control data should be used to enhance of progressively motile sperm, number Klinefelter, S.L. Makris, S.D. Perreault, interpretation of study results. and percent of morphologically normal S. Schrader, D. Seyler, R. Sprando, K.A. Historical data, when used, should be sperm, and number and percent of Treinen, D.N.R. Veeramachaneni, and compiled, presented, and analyzed in an sperm with each identified anomaly. Wise, L.D. Methods for assessing sperm appropriate and relevant manner. In (xi) Stage of the estrous cycle at the motility, morphology, and counts in the order to justify its use as an analytical time of termination for P and F1 rat, rabbit, and dog: a consensus report. tool, information such as the dates of parental females. Reproductive Toxicology. 10(3):237–244 study conduct, the strain and source of (xii) Necropsy findings. (1996). 43838 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(9) Smith, B.J. et al. Comparison of (d) Test procedures—(1) Animal need for individual caging. Animals random and serial sections in selection—(i) Species and strain. should be housed individually in assessment of ovarian toxicity. Testing shall be performed on two dermal studies and during exposure in Reproductive Toxicology. 5:379–383 mammalian species. Rats and mice are inhalation studies. (1991). the species of choice because of their (B) The temperature of the (10) Thomas, J.A. Eds. M.O. Amdur, J. relatively short life spans, limited cost experimental animal rooms should be at Doull, and C.D. Klaassen. Toxic of maintenance, widespread use in 22 ± 3 °C. responses of the reproductive system. pharmacological and toxicological (C) The relative humidity of the Casarett and Doull’s Toxicology studies, susceptibility to tumor experimental animal rooms should be (Pergamon, NY, 1991). induction, and the availability of inbred 30 to 70%. (11) Working, P.K. and Hurtt, M. or sufficiently characterized strains. (D) Where lighting is artificial, the Computerized videomicrographic Commonly used laboratory strains shall sequence should be 12 h light/12 h dark. (E) Control and test animals should be analysis of rat sperm motility. Journal of be used. If other mammalian species are fed from the same batch and lot. The Andrology. 8:330–337 (1987). used, the tester shall provide feed should be analyzed to assure (12) Zenick, H. et al. Ed. Hayes, A.W. justification/reasoning for their uniform distribution and adequacy of Assessment of male reproductive selection. nutritional requirements of the species toxicity: a risk assessment approach. (ii) Age/weight. (A) Testing shall be tested and for impurities that might Principles and Methods of Toxicology started with young healthy animals as influence the outcome of the test. (Raven, NY, 1994). soon as possible after weaning and acclimatization. Animals should be fed and watered ad § 799.9420 TSCA carcinogenicity. (B) Dosing should generally begin no libitum with food replaced at least (a) Scope. This section is intended to later than 8 weeks of age. weekly. meet the testing requirements under (C) At commencement of the study, (F) The study should not be initiated section 4 of TSCA. The objective of a the weight variation of animals used until animals have been allowed a long-term carcinogenicity study is to shall not exceed ± 20% of the mean period of acclimatization/quarantine to observe test animals for a major portion weight for each sex. environmental conditions, nor should of their life span for development of (D) Studies using prenatal or neonatal animals from outside sources be placed neoplastic lesions during or after animals may be recommended under on test without an adequate period of exposure to various doses of a test special conditions. quarantine. (2) Control and test substances. (i) substance by an appropriate route of (iii) Sex. (A) Equal numbers of Where necessary, the test substance is administration. animals of each sex shall be used at dissolved or suspended in a suitable (b) Source. The source material used each dose level. (B) Females shall be nulliparous and vehicle. If a vehicle or diluent is in developing this TSCA test guideline needed, it should not elicit toxic effects is the OPPTS harmonized test guideline nonpregnant. (iv) Numbers. (A) At least 100 rodents itself. It is recommended that wherever 870.4200 (June 1996 Public Draft). This (50 males and 50 females) shall be used possible the use of an aqueous solution source is available at the address in at each dose level and concurrent be considered first, followed by paragraph (g) of this section. control group. consideration of solution in oil, and (c) Definitions. The following (B) If interim sacrifices are planned, finally solution in other vehicles. definitions apply to this section. the number shall be increased by the (ii) One lot of the test substance Carcinogenicity is the development of number of animals scheduled to be should be used, if possible, throughout neoplastic lesions as a result of the sacrificed during the course of the the duration of the study, and the repeated daily exposure of experimental study. research sample should be stored under animals to a chemical by the oral, (C) For a meaningful and valid conditions that maintain its purity and dermal, or inhalation routes of statistical evaluation of long term stability. Prior to the initiation of the exposure. exposure and for a valid interpretation study, there should be a characterization Cumulative toxicity is the adverse of negative results, the number of of the test substance, including the effects of repeated dose occurring as a animals in any group should not fall purity of the test compound, and, if result of prolonged action on, or below 50% at 15 months in mice and 18 possible, the name and quantities of increased concentration of, the months in rats. Survival in any group contaminants and impurities. administered test substance or its should not fall below 25% at 18 months (iii) If the test or control substance is metabolites in susceptible tissues. in mice and 24 months in rats. to be incorporated into feed or another Dose in a carcinogenicity study is the (D) The use of adequate vehicle, the period during which the amount of test substance administered randomization procedures for the test substance is stable in such a via the oral, dermal or inhalation routes proper allocation of animals to test and mixture should be determined prior to for a period of up to 24 months. Dose control groups is required to avoid bias. the initiation of the study. Its is expressed as weight of the test (E) Each animal shall be assigned a homogeneity and concentration should substance (grams, milligrams) per unit unique identification number. Dead be determined prior to the initiation of body weight of test animal (milligram animals, their preserved organs and the study and periodically during the per kilogram), or as weight of the test tissues, and microscopic slides shall be study. Statistically randomized samples substance in parts per million (ppm) in identified by reference to the unique of the mixture should be analyzed to food or drinking water. When exposed numbers assigned. ensure that proper mixing, formulation, via inhalation, dose is expressed as (v) Husbandry. (A) Animals may be and storage procedures are being weight of the test substance per unit group-caged by sex, but the number of followed, and that the appropriate volume of air (milligrams per liter) or as animals per cage must not interfere with concentration of the test or control parts per million. clear observation of each animal. The substance is contained in the mixture. Target organ is any organ of a test biological properties of the test (3) Control groups. A concurrent animal showing evidence of an effect substance or toxic effects (e.g., control group (50 males and 50 females) induced by a test substance. morbidity, excitability) may indicate a is required. This group shall be Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43839 untreated or if a vehicle is used in (3) nonviable (dead) tissues. the test solution should be prepared for administering the test substance, a (4) Anything leading to destruction of different dose levels. With highly toxic vehicle control group. If the toxic the functional integrity of the epidermis substances, the surface area covered properties of the vehicle are not known, (e.g., caking, fissuring, open sores, may be less, but as much of the area as both untreated and vehicle control eschar). possible should be covered with as thin groups are required. (D) Histologic criteria for exceeding and uniform a film as practical. The test (4) Dose levels and dose selection. (i) the high dose: material is not removed after For risk assessment purposes, at least (1) Crust (interfollicular and application. three dose levels shall be used, in follicular). (D) During the exposure period, the (2) Microulcer. addition to the concurrent control application site should not be covered (3) Degeneration/necrosis (mild to group. Dose levels should be spaced to when mice or hamsters are the species produce a gradation of effects. A moderate). (4) Epidermal edema (moderate to of choice. For rats, the test substance rationale for the doses selected must be may be held in contact with the skin provided. marked). (5) Dermal edema (marked). with a porous gauze dressing and (ii) The highest dose level should nonirritating tape if necessary. The test elicit signs of toxicity without (6) Inflammation (marked). (5) Administration of the test site should be further covered in a substantially altering the normal life substance. The three main routes of suitable manner to retain the gauze span due to effects other than tumors. administration are oral, dermal, and dressing and test substance and ensure The highest dose should be determined inhalation. The choice of the route of that the animals cannot ingest the test based on the findings from a 90–day administration depends upon the substance. study to ensure that the dose used is physical and chemical characteristics of (iii) Inhalation studies. (A) The adequate to asses the carcinogenic the test substance and the form animals should be exposed to the test potential of the test substance. Thus, the typifying exposure in humans. substance for 6 h/day on a 7–day per selection of the highest dose to be tested (i) Oral studies. If the test substance week basis, for a period of at least 18 is dependent upon changes observed in is administered by gavage, the animals months in mice and 24 months in rats. several toxicological parameters in are dosed with the test substance on a However, based primarily on practical subchronic studies. The highest dose 7–day per week basis for a period of at considerations, exposure for 6 h/day on tested need not exceed 1,000 mg/kg/day. least 18 months for mice and hamsters a 5–day per week basis is acceptable. (iii) The intermediate-dose level and 24 months for rats. However, based should be spaced to produce a gradation (B) The animals shall be tested in primarily on practical considerations, of toxic effects. dynamic inhalation equipment designed dosing by gavage or via a capsule on a (iv) The lowest dose level should to sustain a minimum air flow of 10 air 5–day per week basis is acceptable. If produce no evidence of toxicity. changes per hr, an adequate oxygen (v) For skin carcinogenicity studies, the test substance is administered in the content of at least 19%, and uniform when toxicity to the skin is a drinking water or mixed in the diet, conditions throughout the exposure determining factor, the highest dose then exposure should be on a 7–day per chamber. Maintenance of slight negative selected should not destroy the week basis. pressure inside the chamber will (ii) Dermal studies. (A) The animals functional integrity of the skin, the prevent leakage of the test substance should be treated with the test intermediate dose should be a into surrounding areas. substance for at least 6 h/day on a 7–day minimally irritating dose, and the low (C) The selection of a dynamic per week basis for a period of at least dose should be the highest nonirritating inhalation chamber should be 18 months for mice and hamsters and 24 dose. appropriate for the test substance and months for rats. However, based (vi) The criteria for selecting the dose test system. Where a whole body primarily on practical considerations, levels for skin carcinogenicity studies, chamber is used to expose animals to an application on a 5–day per week basis based on gross and histopathologic aerosol, individual housing must be is acceptable. Dosing should be dermal lesions, are as follows: used to minimize crowding of the test conducted at approximately the same (A) Gross criteria for reaching the high animals and maximize their exposure to time each day. dose: the test substance. To ensure stability of (B) Fur should be clipped weekly (1) Erythema (moderate). a chamber atmosphere, the total volume from the dorsal area of the trunk of the (2) Scaling. occupied by the test animals shall not test animals. Care should be taken to (3) Edema (mild). exceed 5% of the volume of the test avoid abrading the skin which could (4) Alopecia. chamber. It is recommended, but not alter its permeability. A minimum of 24 (5) Thickening. required, that nose-only or head-only hrs should be allowed for the skin to (B) Histologic criteria for reaching the exposure be used for aerosol studies in recover before the next dosing of the high dose: order to minimize oral exposures due to animal. (1) Epidermal hyperplasia. animals licking compound off their fur. (2) Epidermal hyperkeratosis. (C) The test substance shall be applied uniformly over a shaved area which is Heat stress to the animals should be (3) Epidermal parakeratosis. minimized. (4) Adnexal atrophy/hyperplasia. approximately 10% of the total body (5) Fibrosis. surface area. In order to dose (D) The temperature at which the test approximately 10% of the body surface, is performed should be maintained at (6) Spongiosis (minimal-mild). ± ° (7) Epidermal edema (minimal-mild). the area starting at the scapulae 22 2 C. The relative humidity should (8) Dermal edema (minimal- (shoulders) to the wing of the ileum be maintained between 40 to 60%, but moderate). (hipbone) and half way down the flank in certain instances (e.g., tests of (9) Inflammation (moderate). on each side of the animal should be aerosols, use of water vehicle) this may (C) Gross criteria for exceeding the shaved. The volume of application not be practicable. high dose: should be kept constant and should not (E) The rate of air flow shall be (1) Ulcers, fissures. exceed 100 µL for the mouse and 300 µL monitored continuously but recorded at (2) Exudate/crust (eschar). for the rat; different concentrations of least three times during exposure. 43840 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(F) Temperature and humidity shall should include, but not be limited to, be preserved in a suitable medium for be monitored continuously but should evaluation of skin and fur, eyes and possible future histopathological be recorded at least every 30 minutes. mucous membranes, respiratory and examination. (G) The actual concentrations of the circulatory effects, autonomic effects (A) Digestive system. test substance shall be measured in the such as salivation, central nervous (1) Salivary glands. breathing zone. During the exposure system effects, including tremors and (2) Esophagus. period, the actual concentrations of the convulsions, changes in the level of (3) Stomach. test substance should be held as activity, gait and posture, reactivity to (4) Duodenum. constant as practicable, monitored handling or sensory stimuli, altered (5) Jejunum. continuously or intermittently strength and stereotypes or bizarre (6) Ileum. depending on the method of analysis. behavior (e.g., self-mutilation, walking (7) Cecum. Chamber concentrations may be backwards). (8) Colon. measured using gravimetric or (iii) Body weights shall be recorded (9) Rectum. analytical methods as appropriate. If individually for all animals; once a (10) Liver. trial run measurements are reasonably week during the first 13 weeks of the (11) Pancreas. consistent (± 10% for liquid aerosol, gas, study and at least once every 4 weeks, (12) Gallbladder (mice). or dry aerosol), the two measurements thereafter, unless signs of clinical (13) Bile duct (rat). should be sufficient. If measurements toxicity suggest more frequent weighing (B) Nervous system. are not consistent, then three to four to facilitate monitoring of health status. (1) Brain (multiple sections). measurements should be taken. (iv) When the test substance is (2) Pituitary. (H) During the development of the administered in the feed or drinking (3) Peripheral nerves. generating system, particle size analysis water, measurements of feed or water (4) Spinal cord (three levels). shall be performed to establish the consumption, respectively, should be (5) Eyes (retina, optic nerve). stability of aerosol concentrations with determined weekly during the first 13 (C) Glandular system. (1) Adrenals. respect to particle size. Measurement of weeks of the study and then at (2) Parathyroids. aerodynamic particle size in the approximately monthly intervals unless (3) Thyroids. animals’s breathing zone should be health status or body weight changes (D) Respiratory system. measured during a trial run. If median dictate otherwise. (1) Trachea. (v) Moribund animals shall be aerodynamic diameter (MMAD) values (2) Lung. removed and sacrificed when noticed for each exposure level are within 10% (3) Pharynx. of each other, then two measurements and the time of death should be (4) Larynx. during the exposures should be recorded as precisely as possible. At the (5) Nose (inhalation studies only). sufficient. If pretest measurements are end of the study period, all survivors (E) Cardiovascular/hematopoietic not within 10% of each other, three to shall be sacrificed. system. four measurements should be taken. The (8) Clinical pathology. At 12 months, (1) Aorta (thoracic). MMAD particle size range should be 18 months, and at terminal sacrifice, a (2) Heart. µ between 1–3 m. The particle size of blood smear shall be obtained from all (3) Bone marrow. hygroscopic materials should be small animals. A differential blood count (4) Lymph nodes. enough to allow pulmonary deposition should be performed on blood smears (5) Spleen. once the particles swell in the moist from those animals in the highest (6) Thymus. environment of the respiratory tract. dosage group and the controls from the (F) Urogenital system. (I) Feed shall be withheld during terminal sacrifice. If these data, or data (1) Kidneys. exposure. Water may also be withheld from the pathological examination (2) Urinary bladder. during exposure. indicate a need, then the 12– and 18– (3) Prostate. (6) Observation period. It is necessary month blood smears should also be (4) Testes/epididymides. that the duration of the carcinogenicity examined. Differential blood counts (5) Seminal vesicles. study comprise the majority of the should be performed for the next lower (6) Uterus. normal life span of the strain of animals groups if there is a major discrepancy (7) Ovaries. used. This time period shall not be less between the highest group and the (G) Other. than 24 months for rats and 18 months controls. If clinical observations suggest (1) Lacrimal gland. for mice, and ordinarily not longer than a deterioration in health of the animals (2) Mammary gland. 30 months for rats and 24 months for during the study, a differential blood (3) Skin. mice. For longer time periods, and count of the affected animals shall be (4) Skeletal muscle. where any other species are used, performed. (5) All gross lesions and masses. consultation with the Agency in regard (9) Gross necropsy. (i) A complete (6) Sternum and/or femur. to the duration of the study is advised. gross examination shall be performed on (iv) In inhalation studies, the entire (7) Observation of animals. (i) all animals, including those that died respiratory tract, including nose, Observations shall be made at least once during the experiment or were killed in pharynx, larynx, and paranasal sinuses each day for morbidity and mortality. a moribund condition. should be examined and preserved. In Appropriate actions should be taken to (ii) The liver, lungs, kidneys, brain, dermal studies, skin from treated and minimize loss of animals from the study and gonads should be trimmed and adjacent control skin sites should be (e.g., necropsy or refrigeration of those weighed wet as soon as possible after examined and preserved. animals found dead and isolation or dissection to avoid drying. The organs (v) Inflation of lungs and urinary sacrifice of weak or moribund animals). should be weighed from interim bladder with a fixative is the optimal (ii) A careful clinical examination sacrifice animals as well as from at least method for preservation of these tissues. shall be made at least once weekly. 10 animals per sex per group at terminal The proper inflation and fixation of the Observations should be detailed and sacrifice. lungs in inhalation studies is essential carefully recorded, preferably using (iii) The following organs and tissues, for appropriate and valid explicitly defined scales. Observations or representative samples thereof, shall histopathological examination. Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43841

(vi) Information from clinical target organs, body weight changes, (A) Time of death during the study or pathology, and other in-life data should effects on mortality, and any other whether animals survived to be considered before microscopic general or specific toxic effects. termination. examination, since they may provide (ii) In any study which demonstrates (B) Time of observation of each significant guidance to the pathologist. an absence of toxic effects, further abnormal sign and its subsequent (10) Histopathology. (i) The following investigation to establish absorption and course. histopathology shall be performed: bioavailablity of the test substance (C) Body weight data. (A) Full histopathology on the organs should be considered. (D) Feed and water consumption data, and tissues under paragraph (d)(9) (iii) (iii) In order for a negative test to be when collected. of this section of all animals in the acceptable, it must meet the following (E) Results of clinical pathology and control and high dose groups and all criteria: No more than 10% of any group immunotoxicity screen when animals that died or were killed during is lost due to autolysis, cannibalism, or performed. the study. management problems; and survival in (F) Necropsy findings including (B) All gross lesions in all animals. each group is no less than 50% at 15 absolute/relative organ weight data. (C) Target organs in all animals. months for mice and 18 months for rats. (G) Detailed description of all (D) Lungs, liver, and kidneys of all Survival should not fall below 25% at histopathological findings. animals. Special attention to 18 months for mice and 24 months for (H) Statistical treatment of results examination of the lungs of rodents rats. where appropriate. should be made for evidence of (iv) The use of historical control data (I) Historical control data. infection since this provides an from an appropriate time period from (iii) Inhalation studies. In addition, assessment of the state of health of the the same testing laboratory (i.e., the for inhalation studies the following animals. incidence of tumors and other suspect shall be reported: (ii) If the results show substantial lesions normally occurring under the (A) Test conditions. The following alteration of the animal’s normal life same laboratory conditions and in the exposure conditions shall be reported. span, the induction of effects that might same strain of animals employed in the (1) Description of exposure apparatus affect a neoplastic response, or other test) is helpful for assessing the including design, type, dimensions, effects that might compromise the significance of changes observed in the source of air, system for generating significance of the data, the next lower current study. particulate and aerosols, method of dose levels shall be examined as (3) Test report. (i) In addition to the conditioning air, treatment of exhaust described under paragraph (d)(11)(i) of reporting requirements as specified air and the method of housing the this section. under 40 CFR part 792, subpart J, the animals in a test chamber. (iii) An attempt should be made to following specific information shall be (2) The equipment for measuring correlate gross observations with reported. Both individual and summary temperature, humidity, and particulate microscopic findings. data should be presented. aerosol concentrations and size should (iv) Tissues and organs designated for (A) Test substance characterization be described. microscopic examination should be should include: (B) Exposure data. These shall be fixed in 10% buffered formalin or a (1) Chemical identification. tabulated and presented with mean recognized suitable fixative as soon as (2) Lot or batch number. values and a measure of variability (e.g. necropsy is performed and no less than (3) Physical properties. standard deviation) and should include: 48 hrs prior to trimming. Tissues should (4) Purity/impurities. (1) Airflow rates through the be trimmed to a maximum thickness of (5) Identification and composition of inhalation equipment. 0.4 cm for processing. any vehicle used. (2) Temperature and humidity of air. (e) Data and reporting—(1) Treatment (B) Test system should contain data (3) Actual (analytical or gravimetric) of results. (i) Data shall be summarized on: concentration in the breathing zone. in tabular form, showing for each test (1) Species and strain of animals used (4) Nominal concentration (total group the number of animals at the start and rationale for selection if other than amount of test substance fed into the of the test, the number of animals that recommended. inhalation equipment divided by showing lesions, the types of lesions, (2) Age including body weight data volume of air). and the percentage of animals and sex (5) Particle size distribution, (3) Test environment including cage displaying each type of lesion. calculated MMAD and geometric (ii) All observed results (quantitative conditions, ambient temperature, standard deviation (GSD). and qualitative) shall be evaluated by an humidity, and light/dark periods. (6) Explanation as to why the desired (C) Test procedure should include the appropriate statistical method. Any chamber concentration and/or particle following data: size could not be achieved (if generally accepted statistical methods (1) Method of randomization used. may be used; the statistical methods (2) Full description of experimental applicable) and the efforts taken to including significance criteria shall be design and procedure. comply with this aspect of the sections. selected during the design of the study. (3) Dose regimen including levels, (f) Quality assurance. A system shall (2) Evaluation of study results. (i) The methods, and volume. be developed and maintained to assure findings of a carcinogenicity study (4) Test results—(i) Group animal and document adequate performance of should be evaluated in conjunction with data. Tabulation of toxic response data laboratory staff and equipment. The the findings of previous studies and by species, strain, sex, and exposure study shall be conducted in compliance considered in terms of the toxic effects, level for: with 40 CFR Part 792—Good Laboratory the necropsy and histopathological (A) Number of animals exposed. Practice Standards. findings. The evaluation shall include (B) Number of animals showing signs (g) References. For additional the relationship between the dose of the of toxicity. background information on this test test substance and the presence, (C) Number of animals dying. guideline, the following references incidence, and severity of abnormalities (ii) Individual animal data. Data should be consulted. These references (including behavioral and clinical should be presented as summary (group are available for inspection at the TSCA abnormalities), gross lesions, identified mean) as well as for individual animals. Nonconfidential Information Center, 43842 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Rm. NE–B607, Environmental involved in tumor formation in humans demonstrated that many chemicals that Protection Agency, 401 M St., SW., and experimental animals. The bacterial are positive in this test also exhibit Washington, DC, 12 noon to 4 p.m., reverse mutation test is rapid, mutagenic activity in other tests. There Monday through Friday, except legal inexpensive and relatively easy to are examples of mutagenic agents which holidays. perform. Many of the test strains have are not detected by this test; reasons for (1) Benitz, K.F. Ed. Paget, G.E. several features that make them more these shortcomings can be ascribed to Measurement of Chronic Toxicity. sensitive for the detection of mutations, the specific nature of the endpoint Methods of Toxicology (Blackwell, including responsive DNA sequences at detected, differences in metabolic Oxford, 1970) pp. 82–131. the reversion sites, increased cell activation, or differences in (2) Fitzhugh, O.G. Chronic Oral permeability to large molecules and bioavailability. On the other hand, Toxicity, Appraisal of the Safety of elimination of DNA repair systems or factors which enhance the sensitivity of Chemicals in Foods, Drugs and enhancement of error-prone DNA repair the bacterial reverse mutation test can Cosmetics. The Association of Food and processes. The specificity of the test lead to an overestimation of mutagenic Drug Officials of the United States. pp. strains can provide some useful activity. 36–45 (1959, 3rd Printing 1975). information on the types of mutations (3) The bacterial reverse mutation test (3) Goldenthal, E.I. and D’Aguanno, that are induced by genotoxic agents. A may not be appropriate for the W. Evaluation of Drugs, Appraisal of the very large data base of results for a wide evaluation of certain classes of Safety of Chemicals in Foods, Drugs, variety of structures is available for chemicals, for example highly and Cosmetics. The Association of Food bacterial reverse mutation tests and bactericidal compounds (e.g. certain and Drug Officials of the United States. well-established methodologies have antibiotics) and those which are thought pp. 60–67 (1959, 3rd Printing 1975). been developed for testing chemicals (or known) to interfere specifically with (4) Organisation for Economic Co- with different physico-chemical the mammalian cell replication system operation and Development. Guidelines properties, including volatile (e.g. some topoisomerase inhibitors and for Testing of Chemicals, Section 4– compounds. some nucleoside analogues). In such Health Effects, Part 451 Carcinogenicity (b) Source. The source material used cases, mammalian mutation tests may Studies (Paris, 1981). in developing this TSCA test guideline be more appropriate. (5) Page, N.P. Chronic Toxicity and are the OECD replacement guidelines (4) Although many compounds that Carcinogenicity Guidelines. Journal of for 471 and 472 (February 1997). This are positive in this test are mammalian Environmental Pathology and source is available at the address in carcinogens, the correlation is not Toxicology. 11:161–182 (1977). paragraph (g) of this section. absolute. It is dependent on chemical (6) Page, N.P. Eds. Kraybill and (c) Definitions. The following class and there are carcinogens that are Mehlman. Concepts of a Bioassay definitions apply to this section: not detected by this test because they Program in Environmental A reverse mutation test in either act through other, non-genotoxic Carcinogenesis. Vol.3. Advances in Salmonella typhimurium or Escherichia mechanisms or mechanisms absent in Modern Toxicology (Hemisphere, coli detects mutation in an amino-acid bacterial cells. Washington, DC., 1977) pp. 87–171. requiring strain (histidine or (e) Test method—(1) Principle. (i) (7) Sontag, J.M. et al. Guidelines for tryptophan, respectively) to produce a Suspensions of bacterial cells are Carcinogen Bioassay in Small Rodents. strain independent of an outside supply exposed to the test substance in the NCI-CS-TR-1 United States Cancer of amino-acid. presence and in the absence of an Institute, Division of Cancer Control and Base pair substitution mutagens are exogenous metabolic activation system. Prevention, Carcinogenesis Bioassay agents that cause a base change in DNA. In the plate incorporation method, these Program (Bethesda, MD). In a reversion test this change may suspensions are mixed with an overlay occur at the site of the original agar and plated immediately onto § 799.9510 TSCA bacterial reverse mutation, or at a second site in the minimal medium. In the preincubation mutation test. bacterial genome. method, the treatment mixture is (a) Scope. This section is intended to Frameshift mutagens are agents that incubated and then mixed with an meet the testing requirements under cause the addition or deletion of one or overlay agar before plating onto minimal section 4 of TSCA. more base pairs in the DNA, thus medium. For both techniques, after 2 or (1) The bacterial reverse mutation test changing the reading frame in the RNA 3 days of incubation, revertant colonies uses amino-acid requiring strains of (d) Initial considerations. (1) The are counted and compared to the Salmonella typhimurium and bacterial reverse mutation test utilizes number of spontaneous revertant Escherichia coli to detect point prokaryotic cells, which differ from colonies on solvent control plates. mutations, which involve substitution, mammalian cells in such factors as (ii) Several procedures for performing addition or deletion of one or a few uptake, metabolism, chromosome the bacterial reverse mutation test have DNA base pairs. The principle of this structure and DNA repair processes. been described. Among those commonly bacterial reverse mutation test is that it Tests conducted in vitro generally used are the plate incorporation detects mutations which revert require the use of an exogenous source method, the preincubation method, the mutations present in the test strains and of metabolic activation. In vitro fluctuation method, and the suspension restore the functional capability of the metabolic activation systems cannot method. Suggestions for modifications bacteria to synthesize an essential mimic entirely the mammalian in vivo for the testing of gases or vapors are amino acid. The revertant bacteria are conditions. The test therefore does not described in the reference in paragraph detected by their ability to grow in the provide direct information on the (g)(12) of this section. absence of the amino acid required by mutagenic and carcinogenic potency of (iii) The procedures described in this the parent test strain. a substance in mammals. section pertain primarily to the plate (2) Point mutations are the cause of (2) The bacterial reverse mutation test incorporation and preincubation many human genetic diseases and there is commonly employed as an initial methods. Either of them is acceptable is substantial evidence that point screen for genotoxic activity and, in for conducting experiments both with mutations in oncogenes and tumor particular, for point mutation-inducing and without metabolic activation. Some suppressor genes of somatic cells are activity. An extensive data base has compounds may be detected more Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43843 efficiently using the preincubation (iii) S. typhimurium TA98. chemical being tested. In some cases it method. These compounds belong to (iv) S. typhimurium TA100. may be appropriate to utilize more than chemical classes that include short (v) E. coli WP2 uvrA, or E. coli WP2 one concentration of post-mitochondrial chain aliphatic nitrosamines, divalent uvrA (pKM101), or S. typhimurium fraction. For azo-dyes and diazo- metals, aldehydes, azo-dyes and diazo TA102. In order to detect cross-linking compounds, using a reductive metabolic compounds, pyrollizidine alkaloids, mutagens it may be preferable to activation system may be more allyl compounds and nitro compounds. include TA102 or to add a DNA repair- appropriate (the system described in the It is also recognized that certain classes proficient strain of E.coli [e.g. E.coli references under paragraphs (g)(6) and of mutagens are not always detected WP2 or E.coli WP2 (pKM101).] (g)(13) of this section may be used). using standard procedures such as the (4) Established procedures for stock (D) Test substance/preparation. Solid plate incorporation method or culture preparation, marker verification test substances should be dissolved or preincubation method. These should be and storage should be used. The amino- suspended in appropriate solvents or regarded as ‘‘special cases’’ and it is acid requirement for growth should be vehicles and diluted if appropriate prior strongly recommended that alternative demonstrated for each frozen stock to treatment of the bacteria. Liquid test procedures should be used for their culture preparation (histidine for S. substances may be added directly to the detection. The following ‘‘special cases’’ typhimurium strains, and tryptophan for test systems and/or diluted prior to could be identified (together with E. coli strains). Other phenotypic treatment. Fresh preparations should be examples of procedures that could be characteristics should be similarly employed unless stability data used for their detection): azo-dyes and checked, namely: the presence or demonstrate the acceptability of storage. diazo compounds (alterative procedures absence of R-factor plasmids where (ii) Test conditions—(A) Solvent/ are described in the references in appropriate [i.e. ampicillin resistance in vehicle. The solvent/vehicle shall not be paragraphs (g)(3), (g)(5), (g)(6), and strains TA98, TA100 and TA97a or suspected of chemical reaction with the (g)(13) of this section), gases and volatile TA97, WP2 uvrA and WP2 uvrA test substance and shall be compatible chemicals (alterative procedures are (pKM101), and ampicillin + tetracycline with the survival of the bacteria and the described in the references in resistance in strain TA102]; the S9 activity (see paragraph (g)(22) of this paragraphs (g)(12), (g)(14), (g)(15), and presence of characteristic mutations (i.e. section). If other than well-known (g)(16) of this section), and glycosides rfa mutation in S. typhimurium through solvent/vehicles are used, their (alterative procedures are described in sensitivity to crystal violet, and uvrA inclusion should be supported by data the references in paragraphs (g)(17) and mutation in E. coli or uvrB mutation in indicating their compatibility. It is (g)(18) of this section). A deviation from S. typhimurium, through sensitivity to recommended that wherever possible, the standard procedure needs to be ultra-violet light). The strains should the use of an aqueous solvent/vehicle be scientifically justified. also yield spontaneous revertant colony considered first. When testing water- (2) Description—(i) Preparations—(A) plate counts within the frequency unstable substances, the organic Bacteria. (1) Fresh cultures of bacteria ranges expected from the laboratory’s solvents used should be free of water. should be grown up to the late historical control data and preferably (B) Exposure concentrations. (1) exponential or early stationary phase of within the range reported in the Amongst the criteria to be taken into growth (approximately 109 cells per ml). literature. consideration when determining the Cultures in late stationary phase should (B) Medium. An appropriate minimal highest amount of test substance to be not be used. The cultures used in the agar (e.g. containing Vogel-Bonner used are cytotoxicity and solubility in experiment shall contain a high titre of minimal medium E and glucose) and an the final treatment mixture. It may be viable bacteria. The titre may be overlay agar containing histidine and useful to determine toxicity and demonstrated either from historical biotin or tryptophan, to allow for a few insolubility in a preliminary control data on growth curves, or in cell divisions, shall be used. The experiment. Cytotoxicity may be each assay through the determination of procedures described in the references detected by a reduction in the number viable cell numbers by a plating under paragraphs (g)(1), (g)(2), and (g)(9) of revertant colonies, a clearing or experiment. of this section may be used for this diminution of the background lawn, or (2) The culture temperature shall be analysis. the degree of survival of treated 37°C. (C) Metabolic activation. Bacteria cultures. The cytotoxicity of a substance (3) At least five strains of bacteria shall be exposed to the test substance may be altered in the presence of should be used. These should include both in the presence and absence of an metabolic activation systems. four strains of S. typhimurium (TA1535; appropriate metabolic activation system. Insolubility should be assessed as TA1537 or TA97a or TA97; TA98; and The most commonly used system is a precipitation in the final mixture under TA100) that have been shown to be cofactor-supplemented post- the actual test conditions and evident to reliable and reproducibly responsive mitochondrial fraction (S9) prepared the unaided eye. The recommended between laboratories. These four S. from the livers of rodents treated with maximum test concentration for soluble typhimurium strains have GC base pairs enzyme-inducing agents such as Aroclor non-cytotoxic substances is 5 mg/plate at the primary reversion site and it is 1254 (the system described in the or 5 µl/plate. For non-cytotoxic known that they may not detect certain references under paragraphs (g)(1) and substances that are not soluble at 5mg/ oxidizing mutagens, cross-linking (g)(2) of this section may be used) or a plate or 5µl/plate, one or more agents, and hydrazines. Such substances combination of phenobarbitone and β- concentrations tested should be may be detected by E.coli WP2 strains naphthoflavone (the system described in insoluble in the final treatment mixture. or S. typhimurium TA102 (see the references under paragraphs (g)(18), Test substances that are cytotoxic paragraph (g)(19) of this section) which (g)(20), and (g)(21) of this section may already below 5mg/plate or 5µl/plate have an AT base pair at the primary be used). The post-mitochondrial should be tested up to a cytotoxic reversion site. Therefore the fraction is usually used at concentration. The precipitate should recommended combination of strains is: concentrations in the range from 5 to not interfere with the scoring. (i) S. typhimurium TA1535. 30% v/v in the S9-mix. The choice and (2) At least five different analyzable (ii) S. typhimurium TA1537 or TA97 condition of a metabolic activation concentrations of the test substance or TA97a. system may depend upon the class of shall be used with approximately half 43844 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations log (i.e. √10) intervals between test (C) Controls. (1) Concurrent strain- positive control reference substance(s) points for an initial experiment. Smaller specific positive and negative (solvent should be selected on the basis of the intervals may be appropriate when a or vehicle) controls, both with and type of bacteria strains used. The concentration-response is being without metabolic activation, shall be following chemicals are examples of investigated. included in each assay. Positive control suitable positive controls for assays with (3) Testing above the concentration of concentrations that demonstrate the metabolic activation: 5 mg/plate or 5µl/plate may be effective performance of each assay considered when evaluating substances should be selected. containing substantial amounts of (2)(i) For assays employing a potentially mutagenic impurities. metabolic activation system, the

Chemical CAS No.

9,10-Dimethylanthracene ...... [CAS no. 781±43±1] 7,12-Dimethylbenzanthracene ...... [CAS no. 57±97±6] Congo Red (for the reductive metabolic activation method) ...... [CAS no. 573±58±0] Benzo(a)pyrene ...... [CAS no. 50±32±8] Cyclophosphamide (monohydrate) ...... [CAS no. 50±18±0] [CAS no. 6055±19±2] 2-Aminoanthracene ...... [CAS no. 613±13±8]

(ii) 2-Aminoanthracene should not be requires metabolic activation by (3) For assays performed without used as the sole indicator of the efficacy microsomal enzymes, e.g., metabolic activation system, examples of the S9-mix. If 2-aminoanthracene is benzo(a)pyrene, of strain-specific positive controls are: used, each batch of S9 should also be dimethylbenzanthracene. characterized with a mutagen that

Chemical CAS No. Strain

(a) Sodium azide ...... [CAS no. 26628±22±8] ...... TA1535 and TA100 (b) 2-Nitrofluorene ...... [CAS no. 607±57±8] ...... TA 98 (c) 9-Aminoacridine or ICR 191 ...... [CAS no. 90±45±9] or ...... TA1537, TA97 and TA97a [CAS no. 17070±45±0] ...... (d) Cumene hydroperoxide ...... [CAS no. 80±15±9] ...... TA102 (e) Mitomycin C ...... [CAS no. 50±07±7] ...... WP2 uvrA and TA102 (f) N-Ethyl-N-nitro-N-nitrosoguanidine or [CAS no. 70±25±7] or ...... WP2, WP2 uvrA and WP2 uvrA (pKM101) 4-nitroquinoline 1-oxide ...... [CAS no. 56±57±5] ...... (g) Furylfuramide (AF-2) ...... [CAS no. 3688±53±7] ...... Plasmid-containing strains

(4) Other appropriate positive control metabolic activation mixture containing should be aerated during pre-incubation reference substances may be used. The an adequate amount of post- by using a shaker. use of chemical class-related positive mitochondrial fraction (in the range (C) For an adequate estimate of control chemicals may be considered, from 5 to 30% v/v in the metabolic variation, triplicate plating should be when available. activation mixture) are mixed with the used at each dose level. The use of (5) Negative controls, consisting of overlay agar (2.0 ml), together with the duplicate plating is acceptable when solvent or vehicle alone, without test bacteria and test substance/test solution. scientifically justified. The occasional substance, and otherwise treated in the The contents of each tube are mixed and loss of a plate does not necessarily same way as the treatment groups, shall poured over the surface of a minimal invalidate the assay. be included. In addition, untreated agar plate. The overlay agar is allowed (D) Gaseous or volatile substances controls should also be used unless to solidify before incubation. should be tested by appropriate there are historical control data methods, such as in sealed vessels demonstrating that no deleterious or (B) For the preincubation method the (methods described in the references mutagenic effects are induced by the test substance/test solution is under paragraphs (g)(12), (g)(14), (g)(15), chosen solvent. preincubated with the test strain and (g)(16) of this section may be used). 8 (3) Procedure—(i) Treatment with test (containing approximately 10 viable (ii) Incubation. All plates in a given substance. (A) For the plate cells) and sterile buffer or the metabolic assay shall be incubated at 37 °C for 48– incorporation method, without activation system (0.5 ml) usually for 20 72 hrs. After the incubation period, the ° metabolic activation, usually 0.05 ml or min. or more at 30–37 C prior to mixing number of revertant colonies per plate is 0.1 ml of the test solutions, 0.1 ml of with the overlay agar and pouring onto counted. fresh bacterial culture (containing the surface of a minimal agar plate. (f) Data and reporting—(1) Treatment approximately 108 viable cells) and 0.5 Usually, 0.05 or 0.1 ml of test substance/ of results. (i) Data shall be presented as ml of sterile buffer are mixed with 2.0 test solution, 0.1 ml of bacteria, and 0.5 the number of revertant colonies per ml of overlay agar. For the assay with ml of S9-mix or sterile buffer, are mixed plate. The number of revertant colonies metabolic activation, usually 0.5 ml of with 2.0 ml of overlay agar. Tubes on both negative (solvent control, and Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43845 untreated control if used) and positive reported. Both individual and summary (3) Gatehouse, D., Haworth, S., control plates shall also be given. data should be presented. Cebula, T., Gocke, E., Kier, L., (ii) Individual plate counts, the mean (i) Test substance: Matsushima, T., Melcion, C., Nohmi, T., number of revertant colonies per plate (A) Identification data and CAS no., if Venitt, S., and Zeiger, E. and the standard deviation shall be known. Recommendations for the Performance presented for the test substance and (B) Physical nature and purity. of Bacterial Mutation Assays. Mutation positive and negative (untreated and/or (C) Physicochemical properties Research. 312, 217–233 (1994). solvent) controls. relevant to the conduct of the study. (4) Kier, L.D., Brusick, D.J., Auletta, (iii) There is no requirement for (D) Stability of the test substance, if A.E., Von Halle, E.S., Brown, M.M., verification of a clear positive response. known. Simmon, V.F., Dunkel, V., McCann, J., Equivocal results shall be clarified by (ii) Solvent/vehicle: Mortelmans, K., Prival, M., Rao, T.K., further testing preferably using a (A) Justification for choice of solvent/ and Ray V. The Salmonella modification of experimental vehicle. Typhimurium/Mammalian Microsomal conditions. Negative results need to be (B) Solubility and stability of the test Assay: A Report of the U.S. confirmed on a case-by-case basis. In substance in solvent/vehicle, if known. Environmental Protection Agency Gene- those cases where confirmation of (iii) Strains: Tox Program. Mutation Research. 168, negative results is not considered (A) Strains used. 69–240 (1986). necessary, justification should be (B) Number of cells per culture. (5) Yahagi, T., Degawa, M., Seino, provided. Modification of study (C) Strain characteristics. Y.Y., Matsushima, T., Nagao, M., parameters to extend the range of (iv) Test conditions: Sugimura, T., and Hashimoto, Y. conditions assessed should be (A) Amount of test substance per plate Mutagenicity of Carcinogen Azo Dyes considered in follow-up experiments. (mg/plate or ml/plate) with rationale for and Their Derivatives. Cancer Letters, 1. Study parameters that might be selection of dose and number of plates 91–96 (1975). modified include the concentration per concentration. (6) Matsushima, M., Sugimura, T., spacing, the method of treatment (plate (B) Media used. Nagao, M., Yahagi, T., Shirai, A., and incorporation or liquid preincubation), (C) Type and composition of Sawamura, M. Factors Modulating and metabolic activation conditions. metabolic activation system, including Mutagenicity Microbial Tests. Eds. (2) Evaluation and interpretation of acceptability criteria. Norpoth, K.H. and Garner, R.C. Short- results. (i) There are several criteria for (D) Treatment procedures. Term Test Systems for Detecting determining a positive result, such as a (v) Results: Carcinogens (Springer, Berlin- concentration-related increase over the (A) Signs of toxicity. Heidelberg-New York, 1980) pp. 273– range tested and/or a reproducible (B) Signs of precipitation. 285. increase at one or more concentrations (C) Individual plate counts. (7) Gatehouse, D.G., Rowland, I.R., in the number of revertant colonies per (D) The mean number of revertant Wilcox, P., Callender, R.D., and Foster, plate in at least one strain with or colonies per plate and standard R. Bacterial Mutation Assays. Ed. without metabolic activation system. deviation. Kirkland, D.J. Basic Mutagenicity Tests. Biological relevance of the results (E) Dose-response relationship, where UKEMS Part 1 Revised (Cambridge should be considered first. Statistical possible. University Press, 1990) pp. 13–61. methods may be used as an aid in (F) Statistical analyses, if any. (8) Aeschbacher, H.U., Wolleb, U., evaluating the test results. However, (G) Concurrent negative (solvent/ and Porchet, L.J. Liquid Preincubation statistical significance should not be the vehicle) and positive control data, with Mutagenicity Test for Foods. Food only determining factor for a positive ranges, means and standard deviations. Safety. 8, 167–177 (1987). response. (H) Historical negative (solvent/ (9) Green, M.H.L., Muriel, W.J., and (ii) A test substance for which the vehicle) and positive control data, with Bridges, B.A. Use of a Simplified results do not meet the criteria e.g. ranges, means and standard Fluctuation Test to Detect Low Levels of described under paragraph (f)(2)(i) of deviations. Mutagens. Mutation Research. 38, 33–42 this section is considered non- (vi) Discussion of the results. (1976). mutagenic in this test (vii) Conclusion. (10) Hubbard, S.A., Green, M.H.L., (iii) Although most experiments will (g) References. For additional Gatehouse, D., and J.W. Bridges. The give clearly positive or negative results, background information on this test Fluctuation Test in Bacteria. 2nd in rare cases the data set will preclude guideline, the following references Edition. Ed. Kilbey, B.J., Legator, M., making a definite judgement about the should be consulted. These references Nichols, W., and Ramel C. Handbook of activity of the test substance. Results are available for inspection at the TSCA Mutagenicity Test Procedures (Elsevier, may remain equivocal or questionable Nonconfidential Information Center, Amsterdam-New York-Oxford, 1984) regardless of the number of times the Rm. NE–B607, Environmental pp. 141–161. experiment is repeated. Protection Agency, 401 M St., SW., (11) Thompson, E.D. and Melampy, (iv) Positive results from the bacterial Washington, DC, 12 noon to 4 p.m., P.J. An Examination of the Quantitative reverse mutation test indicate that a Monday through Friday, except legal Suspension Assay for Mutagenesis With substance induces point mutations by holidays. Strains of Salmonella Typhimurium. base substitutions or frameshifts in the (1) Ames, B.N., McCann, J., and Environmental Mutagenesis. 3, 453–465 genome of either Salmonella Yamasaki, E. Methods for Detecting (1981). typhimurium and/or Escherichia coli. Carcinogens and Mutagens With the (12) Araki, A., Noguchi, T., Kato, F., Negative results indicate that under the Salmonella/Mammalian-Microsome and T. Matsushima. Improved Method test conditions, the test substance is not Mutagenicity Test. Mutation Research. for Mutagenicity Testing of Gaseous mutagenic in the tested species. 31, 347–364 (1975). Compounds by Using a Gas Sampling (3) Test report. In addition to the (2) Maron, D.M. and Ames, B.N. Bag. Mutation Research. 307, 335–344 reporting requirements as specified Revised Methods for the Salmonella (1994). under 40 CFR part 792, subpart J, the Mutagenicity Test. Mutation Research. (13) Prival, M.J., Bell, S.J., Mitchell, following specific information shall be 113, 173–215 (1983). V.D., Reipert, M.D., and Vaughn, V.L. 43846 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Mutagenicity of Benzidine and (24) Mahon, G.A.T., Green, M.H.L., Relative total growth is an increase in Benzidine-Congener Dyes and Selected Middleton, B., Mitchell, I., Robinson, cell number over time compared to a Monoazo Dyes in a Modified Salmonella W.D., and Tweats, D.J. Analysis of Data control population of cells; calculated as Assay. Mutation Research. 136, 33–47 from Microbial Colony Assays. UKEMS the product of suspension growth (1984). Sub-Committee on Guidelines for relative to the negative control times (14) Zeiger, E., Anderson, B. E., Mutagenicity Testing Part II. Ed. cloning efficiency relative to negative Haworth, S, Lawlor, T., and Kirkland, D.J. Statistical Evaluation of control. Mortelmans, K. Salmonella Mutagenicity Test Data (Cambridge Survival is the cloning efficiency of Mutagenicity Tests. V. Results from the University Press, 1989) pp. 28–65. the treated cells when plated at the end Testing of 311 Chemicals. Environ. Mol. of the treatment period; survival is Mutagen. 19, 2–141 (1992). § 799.9530 TSCA in vitro mammalian cell usually expressed in relation to the gene mutation test. (15) Simmon, V., Kauhanen, K., and survival of the control cell population. Tardiff, R.G. Mutagenic Activity of (a) Scope. This section is intended to Viability is the cloning efficiency of Chemicals Identified in Drinking Water. meet the testing requirements under the treated cells at the time of plating in Ed. Scott, D., Bridges, B., and Sobels, F. section 4 of TSCA. The in vitro selective conditions after the expression Progress in Genetic Toxicology (Elsevier, mammalian cell gene mutation test can period. Amsterdam, 1977) pp. 249–258. be used to detect gene mutations (d) Initial considerations. (1) In the in (16) Hughes, T.J., Simmons, D.M., induced by chemical substances. vitro mammalian cell gene mutation test, cultures of established cell lines or Monteith, I.G., and Claxton, L.D. Suitable cell lines include L5178Y cell strains can be used. The cells used Vaporization Technique to Measure mouse lymphoma cells, the CHO, AS52 are selected on the basis of growth Mutagenic Activity of Volatile Organic and V79 lines of Chinese hamster cells, ability in culture and stability of the Chemicals in the Ames/Salmonella and TK6 human lymphoblastoid cells spontaneous mutation frequency. Tests Assay. Environmental Mutagenesis. 9, under paragraph (g)(1) of this section. In conducted in vitro generally require the 421–441 (1987). these cell lines the most commonly- use of an exogenous source of metabolic (17) Matsushima, T., Matsumoto, A., used genetic endpoints measure activation. This metabolic activation Shirai, M., Sawamura, M., and mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl system cannot mimic entirely the Sugimura, T. Mutagenicity of the mammalian in vivo conditions. Care Naturally Occurring Carcinogen Cycasin transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl should be taken to avoid conditions and Synthetic Methylazoxy Methane which would lead to results not Conjugates in Salmonella transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different reflecting intrinsic mutagenicity. Typhimurium. Cancer Research. 39, Positive results which do not reflect 3780–3782 (1979). spectra of genetic events. The autosomal location of TK and XPRT may allow the intrinsic mutagenicity may arise from (18) Tamura, G., Gold, C., Ferro-Luzzi, detection of genetic events (e.g. large changes in pH, osmolality or high levels A., and Ames. B.N. Fecalase: A Model deletions) not detected at the HPRT of cytotoxicity. for Activation of Dietary Glycosides to locus on X-chromosomes (For a (2) This test is used to screen for Mutagens by Intestinal Flora. Proc. discussion see the references in possible mammalian mutagens and National Academy of Science. (USA, paragraphs (g)(2), (g)(3), (g)(4),(g)(5), and carcinogens. Many compounds that are 1980) 77, 4961–4965. (g)(6) of this section). positive in this test are mammalian (19) Wilcox, P., Naidoo, A., Wedd, D. (b) Source. The source material used carcinogens; however, there is not a J., and Gatehouse, D. G. Comparison of in developing this TSCA test guideline perfect correlation between this test and Salmonella Typhimurium TA 102 With is the OECD guideline 476 (February carcinogenicity. Correlation is Escherichia Coli WP2 Tester Strains. 1997). This source is available at the dependent on chemical class and there Mutagenesis. 5, 285–291 (1990). address in paragraph (g) of this section. is increasing evidence that there are (20) Matsushima, T., Sawamura, M., (c) Definitions. The following carcinogens that are not detected by this Hara, K., and Sugimura, T. A Safe definitions apply to this section: test because they appear to act through Substitute for Polychlorinated Base pair substitution mutagens are other, non-genotoxic mechanisms or Biphenyls as an Inducer of Metabolic substances which cause substitution of mechanisms absent in bacterial cells. Activation Systems. Ed. F.J. de Serres et one or several base pairs in the DNA. (e) Test method—(1) Principle. (i) al. In Vitro Metabolic Activation in Forward mutation is a gene mutation Cells deficient in thymidine kinase (TK) Mutagenesis Testing. (Elsevier, North from the parental type to the mutant due to the mutation TK∂/- -≤ TK-/- are Holland, 1976) pp. 85–88. form which gives rise to an alteration or resistant to the cytotoxic effects of the (21) Elliott, B.M., Combes, R.D., a loss of the enzymatic activity or the pyrimidine analogue trifluorothymidine Elcombe, C.R., Gatehouse, D.G., Gibson, function of the encoded protein. (TFT). Thymidine kinase proficient cells G.G., Mackay, J.M., and Wolf, R.C. Frameshift mutagens are substances are sensitive to TFT, which causes the Alternatives to Aroclor 1254-Induced S9 which cause the addition or deletion of inhibition of cellular metabolism and in In Vitro Genotoxicity Assays. single or multiple base pairs in the DNA halts further cell division. Thus mutant Mutagenesis. 7, 175–177 (1992). molecule. cells are able to proliferate in the (22) Maron, D., Katzenellenbogen, J., Mutant frequency is the number of presence of TFT, whereas normal cells, and Ames, B.N. Compatibility of mutant cells observed divided by the which contain thymidine kinase, are Organic Solvents With the Salmonella/ number of viable cells. not. Similarly, cells deficient in HPRT Microsome Test. Mutation Research. 88, Phenotypic expression time is a or XPRT are selected by resistance to 6- 343–350 (1981). period during which unaltered gene thioguanine (TG) or 8-azaguanine (AG). (23) Claxton, L.D., Allen, J., Auletta, products are depleted from newly The properties of the test substance A., Mortelmans, K., Nestmann, E., and mutated cells. should be considered carefully if a base Zeiger, E. Guide for the Salmonella Relative suspension growth is an analogue or a compound related to the Typhimurium/Mammalian Microsome increase in cell number over the selective agent is tested in any of the Tests for Bacterial Mutagenicity. expression period relative to the mammalian cell gene mutation tests. For Mutation Research. 189, 83–91 (1987). negative control. example, any suspected selective Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43847 toxicity by the test substance for mutant (B) Media and culture conditions. recommended that wherever possible, and non-mutant cells should be Appropriate culture media and the use of an aqueous solvent/vehicle be investigated. Thus, performance of the incubation conditions (culture vessels, considered first. When testing water- selection system/agent shall be temperature, CO2 concentration and unstable substances, the organic confirmed when testing chemicals humidity) shall be used. Media should solvents used should be free of water. structurally related to the selective be chosen according to the selective Water can be removed by adding a agent. systems and cell type used in the test. molecular sieve. (ii) Cells in suspension or monolayer It is particularly important that culture culture shall be exposed to the test conditions should be chosen that ensure (B) Exposure concentrations. (1) substance, both with and without optimal growth of cells during the Among the criteria to be considered metabolic activation, for a suitable expression period and colony forming when determining the highest period of time and subcultured to ability of both mutant and non-mutant concentration are cytotoxicity and determine cytotoxicity and to allow cells. solubility in the test system and changes phenotypic expression prior to mutant (C) Preparation of cultures. Cells are in pH or osmolality. selection. Cytotoxicity is usually propagated from stock cultures, seeded (2) Cytotoxicity should be determined determined by measuring the relative in culture medium and incubated at with and without metabolic activation ° cloning efficiency (survival) or relative 37 C. Prior to use in this test, cultures in the main experiment using an total growth of the cultures after the may need to be cleansed of pre-existing appropriate indicator of cell integrity treatment period. The treated cultures mutant cells. and growth, such as relative cloning shall be maintained in growth medium (D) Metabolic activation. Cells shall efficiency (survival) or relative total for a sufficient period of time, be exposed to the test substance both in growth. It may be useful to determine characteristic of each selected locus and the presence and absence of an cytotoxicity and solubility in a cell type, to allow near-optimal appropriate metabolic activation system. preliminary experiment. phenotypic expression of induced The most commonly used system is a mutations. Mutant frequency is co-factor-supplemented post- (3) At least four analyzable determined by seeding known numbers mitochondrial fraction (S9) prepared concentrations shall be used. Where of cells in medium containing the from the livers of rodents treated with there is cytotoxicity, these selective agent to detect mutant cells, enzyme-inducing agents such as Aroclor concentrations shall cover a range from and in medium without selective agent 1254 or a combination of the maximum to little or no toxicity; to determine the cloning efficiency phenobarbitone and β-naphthoflavone. this will usually mean that the (viability). After a suitable incubation The post-mitochondrial fraction is concentration levels should be time, colonies shall be counted. The usually used at concentrations in the separated by no more than a factor mutant frequency is derived from the range from 1–10% v/v in the final test between 2 and √10. If the maximum number of mutant colonies in selective medium. The choice and condition of a concentration is based on cytotoxicity medium and the number of colonies in metabolic activation system may then it shall result in approximately 10– non-selective medium. depend upon the class of chemical 20% but not less than 10% relative (2) Description—(i) Preparations—(A) being tested. In some cases it may be survival (relative cloning efficiency) or Cells. (1) A variety of cell types are appropriate to utilize more than one relative total growth. For relatively non- available for use in this test including concentration of post-mitochondrial cytotoxic compounds the maximum subclones of L5178Y, CHO, CHO-AS52, fraction. A number of developments, concentration should be 5 mg/ml, 5 µl/ V79, or TK6 cells. Cell types used in including the construction of genetically ml, or 0.01 M, whichever is the lowest. this test should have a demonstrated engineered cell lines expressing specific sensitivity to chemical mutagens, a high activating enzymes, may provide the (4) Relatively insoluble substances cloning efficiency and a stable potential for endogenous activation. The should be tested up to or beyond their spontaneous mutant frequency. Cells choice of the cell lines used should be limit of solubility under culture should be checked for mycoplasma scientifically justified (e.g. by the conditions. Evidence of insolubility contamination and should not be used relevance of the cytochrome P450 should be determined in the final if contaminated. isoenzyme to the metabolism of the test treatment medium to which cells are (2) The test should be designed to substance). exposed. It may be useful to assess have a predetermined sensitivity and (E) Test substance/preparations. Solid solubility at the beginning and end of power. The number of cells, cultures, test substances should be dissolved or the treatment, as solubility can change and concentrations of test substance suspended in appropriate solvents or during the course of exposure in the test used should reflect these defined vehicles and diluted if appropriate prior system due to presence of cells, S9, parameters. The parameters discussed to treatment of the cells. Liquid test serum etc. Insolubility can be detected in the reference under paragraph (g)(13) substances may be added directly to the by using the unaided eye. The of this section may be used. The test systems and/or diluted prior to precipitate should not interfere with the minimal number of viable cells treatment. Fresh preparations should be scoring. surviving treatment and used at each employed unless stability data (C) Controls. (1) Concurrent positive stage in the test should be based on the demonstrate the acceptability of storage. and negative (solvent or vehicle) spontaneous mutation frequency. A (ii) Test conditions—(A) Solvent/ controls both with and without general guide is to use a cell number vehicle. The solvent/vehicle shall not be metabolic activation shall be included which is at least ten times the inverse suspected of chemical reaction with the in each experiment. When metabolic of the spontaneous mutation frequency. test substance and shall be compatible activation is used the positive control However, it is recommended to utilize with the survival of the cells and the S9 at least 106 cells. Adequate historical activity. If other than well-known chemical shall be one that requires data on the cell system used should be solvent/vehicles are used, their activation to give a mutagenic response. available to indicate consistent inclusion should be supported by data (2) Examples of positive control performance of the test. indicating their compatibility. It is substances include: 43848 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Metabolic Activation condi- tion Locus Chemical CAS No.

Absence of exogenous met- HPRT ...... Ethylmethanesulfonate ...... [CAS no. 62±50±0] abolic activation Ethylnitrosourea ...... [CAS no. 759±73±9] TK (small and large colo- Methylmethanesulfonate ...... [CAS no. 66±27±3] nies). XPRT ...... Ethylmethanesulfonate ...... [CAS no. 62±50±0] Ethylnitrosourea ...... [CAS no. 759±73±9] Presence of exogenous HPRT ...... 3-Methylcholanthrene ...... [CAS no. 56±49±5] metabolic activation. N-Nitrosodimethylamine ...... [CAS no. 62±75±9] 7,12-Dimethylbenzanthracene ...... [CAS no. 57±97±6] TK (small and large colo- Cyclophosphamide (monohydrate) ...... [CAS no. 50±18±0] nies). [CAS no. 6055±19±2] Benzo(a)pyrene ...... [CAS no. 50±32±8] 3-Methylcholanthrene ...... [CAS no. 56±49±5] XPRT ...... N-Nitrosodimethylamine (for high levels of S-9) ...... [CAS no. 62±75±9] Benzo(a)pyrene ...... [CAS no. 50±32±8]

(3) Other appropriate positive control the mutant phenotype. Measurement of (g)(23) of this section. In the TK∂/- test, reference substances may be used, e.g., cytotoxicity by determining the relative colonies are scored using the criteria of if a laboratory has a historical data base cloning efficiency (survival) or relative normal growth (large) and slow growth on 5-Bromo 2′-deoxyuridine [CAS No. total growth of the cultures is usually (small) colonies (a scoring system 59–14–3], this reference substance could initiated after the treatment period. similar to the one described in the be used as well. The use of chemical (B) Each locus has a defined reference under paragraph (g)(24) of this class-related positive control chemicals minimum time requirement to allow section may be used). Mutant cells that may be considered, when available. near optimal phenotypic expression of have suffered the most extensive genetic (4) Negative controls, consisting of newly induced mutants (HPRT and damage have prolonged doubling times solvent or vehicle alone in the treatment XPRT require at least 6–8 days, and TK and thus form small colonies. This medium, and treated in the same way as at least 2 days). Cells are grown in damage typically ranges in scale from the treatment groups shall be included. medium with and without selective the losses of the entire gene to In addition, untreated controls should agent(s) for determination of numbers of karyotypically visible chromosome also be used unless there are historical mutants and cloning efficiency, aberrations. The induction of small control data demonstrating that no respectively. The measurement of colony mutants has been associated deleterious or mutagenic effects are viability (used to calculate mutant with chemicals that induce gross induced by the chosen solvent. frequency) is initiated at the end of the chromosome aberrations. Less seriously (3) Procedure—(i) Treatment with test expression time by plating in non- affected mutant cells grow at rates substance. (A) Proliferating cells shall selective medium. similar to the parental cells and form be exposed to the test substance both (C) If the test substance is positive in large colonies. ∂ - with and without metabolic activation. the L5178Y TK / test, colony sizing (ii) Survival (relative cloning Exposure shall be for a suitable period should be performed on at least one of efficiencies) or relative total growth of time (usually 3 to 6 hrs is effective). the test cultures (the highest positive shall be given. Mutant frequency shall Exposure time may be extended over concentration) and on the negative and be expressed as number of mutant cells one or more cell cycles. positive controls. If the test substance is per number of surviving cells. (B) Either duplicate or single treated negative in the L5178Y TK∂/- test, cultures may be used at each colony sizing should be performed on (iii) Individual culture data shall be concentration tested. When single the negative and positive controls. In provided. Additionally, all data shall be cultures are used, the number of studies using TK6TK∂/-, colony sizing summarized in tabular form. concentrations should be increased to may also be performed. (iv) There is no requirement for ensure an adequate number of cultures (f) Data and reporting—(1) Treatment verification of a clear positive response. for analysis (e.g. at least eight of results. (i) Data shall include Equivocal results shall be clarified by analyzsable concentrations). Duplicate cytotoxicity and viability determination, further testing preferably using a negative (solvent) control cultures colony counts and mutant frequencies modification of experimental should be used. for the treated and control cultures. In conditions. Negative results need to be (C) Gaseous or volatile substances the case of a positive response in the confirmed on a case-by-case basis. In should be tested by appropriate L5178Y TK∂/- test, colonies are scored those cases where confirmation of methods, such as in sealed culture using the criteria of small and large negative results is not considered vessels. Methods described in the colonies on at least one concentration of necessary, justification should be references under paragraphs (g)(20) and the test substance (highest positive provided. Modification of study (g)(21) of this section may be used. concentration) and on the negative and parameters to extend the range of (ii) Measurement of survival, viability, positive control. The molecular and conditions assessed should be and mutant frequency. (A) At the end of cytogenetic nature of both large and considered in follow-up experiments for the exposure period, cells shall be small colony mutants has been explored either equivocal or negative results. washed and cultured to determine in detail and is discussed in the Study parameters that might be survival and to allow for expression of references under paragraphs (g)(22) and modified include the concentration Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43849 spacing, and the metabolic activation (E) Incubation temperature. (4) Aaron, C.S. and Stankowski, Jr., conditions. (F) Incubation time. L.F. Comparison of the AS52/XPRT and (2) Evaluation and interpretation of (G) Duration of treatment. the CHO/HPRT Assays: Evaluation of results. (i) There are several criteria for (H) Cell density during treatment. Six Drug Candidates. Mutation determining a positive result, such as a (I) Type and composition of metabolic Research. 223, 121–128 (1989). concentration-related, or a reproducible activation system including (5) Aaron, C.S., Bolcsfoldi, G., Glatt, increase in mutant frequency. Biological acceptability criteria. H.R., Moore, M., Nishi, Y., Stankowski, relevance of the results should be (J) Positive and negative controls. L., Theiss, J., and Thompson, E. considered first. Statistical methods (K) Length of expression period Mammalian Cell Gene Mutation Assays may be used as an aid in evaluating the (including number of cells seeded, and Working Group Report. Report of the test results. Statistical significance subcultures and feeding schedules, if International Workshop on should not be the only determining appropriate). Standardization of Genotoxicity Test (L) Selective agent(s). factor for a positive response. Procedures. Mutation Research. 312, (M) Criteria for considering tests as (ii) A test substance, for which the 235–239 (1994). positive, negative or equivocal. (6) Scott, D., Galloway, S.M., results do not meet the criteria (N) Methods used to enumerate described in paragraph (f)(2)(i) of this Marshall, R.R., Ishidate, M., Brusick, D., numbers of viable and mutant cells. Ashby, J., and Myhr, B.C. Genotoxicity section is considered non-mutagenic in (O) Definition of colonies of which this system. Under Extreme Culture Conditions. A size and type are considered (including report from ICPEMC Task Group 9. (iii) Although most studies will give criteria for ‘‘small’’ and ‘‘large’’ clearly positive or negative results, in Mutation Research. 257, 147–204 colonies, as appropriate). (1991). rare cases the data set will preclude (v) Results: making a definite judgement about the (7) Clive, D., McCuen, R., Spector, (A) Signs of toxicity. J.F.S., Piper, C., and Mavournin, K.H. activity of the test substance. Results (B) Signs of precipitation. may remain equivocal or questionable (C) Data on pH and osmolality during Specific Gene Mutations in L5178Y regardless of the number of times the the exposure to the test substance, if Cells in Culture. A Report of the U.S. experiment is repeated. determined. Environmental Protection Agency Gene- (iv) Positive results for an in vitro (D) Colony size if scored for at least Tox Program. Mutation Research. 115, mammalian cell gene mutation test negative and positive controls. 225–251 (1983). indicate that the test substance induces (E) Laboratory’s adequacy to detect (8) Li, A.P., Gupta, R.S., Heflich, R.H., gene mutations in the cultured small colony mutants with the L5178Y and Wasson, J. S. A Review and mammalian cells used. A positive TK∂/- system, where appropriate. Analysis of the Chinese Hamster Ovary/ concentration-response that is (F) Dose-response relationship, where Hypoxanthine Guanine Phosphoribosyl reproducible is most meaningful. possible. Transferase System to Determine the Negative results indicate that, under the (G) Statistical analyses, if any. Mutagenicity of Chemical Agents: A test conditions, the test substance does (H) Concurrent negative (solvent/ Report of Phase III of the U.S. not induce gene mutations in the vehicle) and positive control data. Environmental Protection Agency Gene- cultured mammalian cells used. (I) Historical negative (solvent/ Tox Program. Mutation Research. 196, (3) Test report. The test report shall vehicle) and positive control data with 17–36 (1988). include the following information: ranges, means, and standard deviations. (9) Li, A.P., Carver, J.H., Choy, W.N., (i) Test substance: (J) Mutant frequency. Hsie, A.W., Gupta, R.S., Loveday, K.S., (A) Identification data and CAS no., if (vi) Discussion of the results. O’Neill, J.P., Riddle, J.C., Stankowski, known. (vii) Conclusion. Jr., L.F., and Yang, L.L. A Guide for the (B) Physical nature and purity. (g) References. For additional Performance of the Chinese Hamster (C) Physicochemical properties background information on this test Ovary Cell/Hypoxanthine-Guanine relevant to the conduct of the study. guideline, the following references Phosphoribosyl Transferase Gene (D) Stability of the test substance. should be consulted. These references Mutation Assay. Mutation Research. (ii) Solvent/vehicle: are available for inspection at the TSCA 189, 135–141 (1987). (A) Justification for choice of vehicle/ Nonconfidential Information Center, (10) Liber, H.L., Yandell, D.W., and solvent. Rm. NE–B607, Environmental Little, J.B. A Comparison of Mutation (B) Solubility and stability of the test Protection Agency, 401 M St., SW., Induction at the tk and hprt Loci in substance in solvent/vehicle, if known. Washington, DC, 12 noon to 4 p.m., Human Lymphoblastoid Cells; (iii) Cells: Monday through Friday, except legal Quantitative Differences are Due to an (A) Type and source of cells. holidays. Additional Class of Mutations at the (B) Number of cell cultures. (1) Chu, E.H.Y. and Malling, H.V. Autosomal TK Locus. Mutation (C) Number of cell passages, if Mammalian Cell Genetics. II. Chemical Research. 216, 9–17 (1989). applicable. Induction of Specific Locus Mutations (11) Stankowski, L.F. Jr., Tindall, (D) Methods for maintenance of cell in Chinese Hamster Cells In Vitro, Proc. K.R., and Hsie, A.W. Quantitative and cultures, if applicable. National Academy Science (USA, 1968) Molecular Analyses of Ethyl (E) Absence of mycoplasma. 61, 1306–1312. Methanesulfonate- and ICR 191-Induced (iv) Test conditions: (2) Liber, H.L. and Thilly, W.G. Molecular Analyses of Ethyl (A) Rationale for selection of Mutation Assay at the Thymidine Methanesulfonate- and ICR 191-Induced concentrations and number of cell Kinase Locus in Diploid Human Mutation in AS52 Cells. Mutation cultures including e.g., cytotoxicity data Lymphoblasts. Mutation Research. 94, Reseach. 160, 133–147 (1986). and solubility limitations, if available. 467–485 (1982). (12) Turner, N.T., Batson, A.G., and (B) Composition of media, CO2 (3) Moore, M.M., Harrington-Brock, Clive, D. Eds. Kilbey, B.J. et al. concentration. K., Doerr, C.L., and Dearfield, K.L. Procedures for the L5178Y/TK∂/- > (C) Concentration of test substance. Differential Mutant Quantitation at the TK∂/- Mouse Lymphoma Cell (D) Volume of vehicle and test Mouse Lymphoma TK and CHO HGPRT Mutagenicity Assay. Handbook of substance added. Loci. Mutagenesis. 4, 394–403 (1989). Mutagenicity Test Procedures (Elsevier 43850 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Science Publishers, New York, 1984) National Academy Science (USA, 1990) result is chromosomes with pp. 239–268. 87, 51–55. 2,4,8,...chromatids. (13) Arlett, C.F., Smith, D.M., Clarke, (23) Moore, M.M., Clive, D., Hozier, Gap is an achromatic lesion smaller G.M., Green, M.H.L., Cole, J., McGregor, J.C., Howard, B.E., Batson, A.G., Turner, than the width of one chromatid, and D.B., and Asquith, J.C. Ed. Kirkland, D.J. N.T., and Sawyer, J. Analysis of with minimum misalignment of the Mammalian Cell Gene Mutation Assays Trifluorothymidine-Resistant (TFTr) chromatids. Based Upon Colony Formation. Mutants of L5178Y/TK∂/- Mouse Numerical aberration is a change in Statistical Evaluation of Mutagenicity Lymphoma Cells. Mutation Research. the number of chromosomes from the Test Data (Cambridge University Press, 151, 161–174 (1985). normal number characteristic of the 1989) pp. 66–101. (24) Yandell, D.W., Dryja, T.P., and animals utilized. (14) Abbondandolo, A., Bonatti, S., Little J.B. Molecular Genetic Analysis of Polyploidy is a multiple of the Corti, G., Fiorio, R., Loprieno, N., and Recessive Mutations at a Heterozygous haploid chromosome number (n) other Mazzaccaro, A. Induction of 6- Autosomal Locus in Human Cells. than the diploid number (i.e., 3n, 4n Thioguanine-Resistant Mutants in V79 Mutation Research. 229, 89–102 (1990). and so on). Chinese Hamster Cells by Mouse-Liver (25) Moore, M.M. and Doerr, C.L. Structural aberration is a change in Microsome-Activated Comparison of Chromosome Aberration chromosome structure detectable by Dimethylnitrosamine. Mutation Frequency and Small-Colony TK- microscopic examination of the Research. 46, 365–373 (1977). Deficient Mutant Frequency in L5178Y/ metaphase stage of cell division, (15) Ames, B.N., McCann, J., and TK∂/- 3.7.2C Mouse Lymphoma Cells. observed as deletions and fragments, Yamasaki, E. Methods for Detecting Mutagenesis. 5, 609–614 (1990). intrachanges or interchanges. (d) Initial considerations. (1) Rodents Carcinogens and Mutagens with the § 799.9538 TSCA mammalian bone marrow are routinely used in this test. Bone Salmonella/Mammalian-Microsome chromosomal aberration test. marrow is the target tissue in this test, Mutagenicity Test. Mutation Reseach. (a) Scope. This section is intended to since it is a highly vascularised tissue, 31, 347–364 (1975). meet the testing requirements under and it contains a population of rapidly (16) Clive, D., Johnson, K.O., Spector, section 4 of TSCA. The mammalian cycling cells that can be readily isolated J.F.S., Batson, A.G., and Brown M.M.M. bone marrow chromosomal aberration and processed. Other species and target Validation and Characterization of the test is used for the detection of tissues are not the subject of this L5178Y/TK∂/- Mouse Lymphoma structural chromosome aberrations section. Mutagen Assay System. Mutation induced by test compounds in bone (2) This chromosome aberration test is Reseach. 59, 61–108 (1979). marrow cells of animals, usually especially relevant to assessing (17) Maron, D.M. and Ames, B.N. rodents. Structural chromosome mutagenic hazard in that it allows Revised Methods for the Salmonella aberrations may be of two types, consideration of factors of in vivo Mutagenicity Test. Mutation Reseach. chromosome or chromatid. An increase metabolism, pharmacokinetics and 113, 173, 215 (1983). in polyploidy may indicate that a DNA-repair processes although these (18) Elliott, B.M., Combes, R.D., chemical has the potential to induce may vary among species and among Elcombe, C.R., Gatehouse, D.G., Gibson, numerical aberrations. With the tissues. An in vivo test is also useful for G.G., Mackay, J.M., and Wolf, R.C. majority of chemical mutagens, induced further investigation of a mutagenic Alternatives to Aroclor 1254-Induced S9 aberrations are of the chromatid-type, effect detected by an in vitro test. in In Vitro Genotoxicity Assays. but chromosome-type aberrations also (3) If there is evidence that the test Mutagenesis. 7, 175–177 (1992). occur. Chromosome mutations and substance, or a reactive metabolite, will (19) Matsushima, T., Sawamura, M., related events are the cause of many not reach the target tissue, it is not Hara, K., and Sugimura, T. A Safe human genetic diseases and there is appropriate to use this test. Substitute for Polychlorinated substantial evidence that chromosome (e) Test method—(1) Principle. Biphenyls as an Inducer of Metabolic mutations and related events causing Animals are exposed to the test Activation Systems. (Eds.) de Serres, alterations in oncogenes and tumor substance by an appropriate route of F.J., Fouts, J.R., Bend, J.R., and Philpot, suppressor genes are involved in cancer exposure and are sacrificed at R.M. In Vitro Metabolic Activation in in humans and experimental systems. appropriate times after treatment. Prior Mutagenesis Testing (Elsevier, North- (b) Source. The source material used to sacrifice, animals are treated with a Holland, 1976) pp. 85–88. in developing this TSCA test guideline metaphase-arresting agent (e.g., (20) Krahn, D.F., Barsky, F.C., and is the OECD guideline 475 (February colchicine or Colcemid). Chromosome McCooey, K.T. Eds. Tice, R.R., Costa, 1997). This source is available at the preparations are then made from the D.L., and Schaich, K.M. CHO/HGPRT address in paragraph (g) of this section. bone marrow cells and stained, and Mutation Assay: Evaluation of Gases (c) Definitions. The following metaphase cells are analyzed for and Volatile Liquids. Genotoxic Effects definitions apply to this section: chromosome aberrations. of Airborne Agents (New York, Plenum, Chromatid-type aberration is (2) Description—(i) Preparations—(A) 1982) pp. 91–103. structural chromosome damage Selection of animal species. Rats, mice (21) Zamora, P.O., Benson, J.M., Li, expressed as breakage of single and Chinese hamsters are commonly A.P., and Brooks, A.L. Evaluation of an chromatids or breakage and reunion used, although any appropriate Exposure System Using Cells Grown on between chromatids. mammalian species may be used. Collagen Gels for Detecting Highly Chromosome-type aberration is Commonly used laboratory strains of Volatile Mutagens in the CHO/HGPRT structural chromosome damage young healthy adult animals should be Mutation Assay. Environmental expressed as breakage, or breakage and employed. At the commencement of the Mutagenesis. 5, 795–801 (1983). reunion, of both chromatids at an study, the weight variation of animals (22) Applegate, M.L., Moore, M.M., identical site. should be minimal and not exceed ± Broder, C.B., Burrell, A., and Hozier, J.C. Endoreduplication is a process in 20% of the mean weight of each sex. Molecular Dissection of Mutations at the which after an S period of DNA (B) Housing and feeding conditions. Heterozygous Thymidine Kinase Locus replication, the nucleus does not go into The temperature in the experimental in Mouse Lymphoma Cells. Proc. mitosis but starts another S period. The animal room should be 22°C (± 3°C). Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43851

Although the relative humidity should the laboratory conditions for at least 5 (B) Controls. (1) Concurrent positive be at least 30% and preferably not days. and negative (solvent/vehicle) controls exceed 70% other than during room (D) Preparation of doses. Solid test shall be included for each sex in each cleaning, the aim should be 50–60%. substances shall be dissolved or test. Except for treatment with the test Lighting should be artificial, the suspended in appropriate solvents or substance, animals in the control groups sequence being 12 hrs light, 12 hrs dark. vehicles and diluted, as appropriate, should be handled in an identical For feeding, conventional laboratory prior to dosing of the animals. Liquid manner to the animals in the treated diets may be used with an unlimited test substances may be dosed directly or groups. diluted prior to dosing. Fresh supply of drinking water. The choice of preparations of the test substance (2) Positive controls shall produce diet may be influenced by the need to should be employed unless stability structural chromosome aberrations in ensure a suitable admixture of a test data demonstrate the acceptability of vivo at exposure levels expected to give substance when administered by this storage. a detectable increase over background. method. Animals may be housed (ii) Test conditions—(A) Solvent/ Positive control doses should be chosen individually, or be caged in small vehicle. The solvent/vehicle shall not so that the effects are clear but do not groups of the same sex. produce toxic effects at the dose levels immediately reveal the identity of the (C) Preparation of the animals. used, and shall not be suspected of coded slides to the reader. It is Healthy young adult animals shall be chemical reaction with the test acceptable that the positive control be randomly assigned to the control and substance. If other than well-known administered by a route different from treatment groups. Cages should be solvents/vehicles are used, their the test substance and sampled at only a single time. The use of chemical class arranged in such a way that possible inclusion should be supported with data related positive control chemicals may effects due to cage placement are indicating their compatibility. It is recommended that wherever possible, be considered, when available. minimized. The animals are identified the use of an aqueous solvent/vehicle Examples of positive control substances uniquely. The animals are acclimated to should be considered first. include:

Chemical CAS No.

Triethylenemelamine ...... [CAS no. 51±18±3] Ethyl methanesulphonate ...... [CAS no. 62±50±0] Ethyl nitrosourea ...... [CAS no. 759±73±9] Mitomycin C ...... [CAS no. 50±07±7] Cyclophosphamide (monohydrate) ...... [CAS no. 50±18±0] [CAS no. 6055±19±2]

(3) Negative controls, treated with be performed with animals of the arresting agent (e.g. Colcemid or solvent or vehicle alone, and otherwise appropriate sex. colchicine). Animals are sampled at an treated in the same way as the treatment (ii) Treatment schedule. (A) Test appropriate interval thereafter. For mice groups, shall be included for every substances are preferably administered this interval is approximately 3–5 hrs; sampling time, unless acceptable inter- as a single treatment. Test substances for Chinese hamsters this interval is animal variability and frequencies of may also be administered as a split approximately 4–5 hrs. Cells shall be cells with chromosome aberrations are dose, i.e. two treatments on the same harvested from the bone marrow and available from historical control data. If day separated by no more than a few analyzed from chromosome aberrations. single sampling is applied for negative hrs, to facilitate administering a large (iii) Dose levels. If a range finding controls, the most appropriate time is volume of material. Other dose regimens study is performed because there are no the first sampling time. In the absence should be scientifically justified. suitable data available, it shall be of historical or published control data (B) Samples shall be taken at two performed in the same laboratory, using demonstrating that no deleterious or separate times following treatment on the same species, strain, sex, and mutagenic effects are induced by the one day. For rodents, the first sampling treatment regimen to be used in the chosen solvent/vehicle, untreated interval is 1.5 normal cell cycle length main study (an approach to dose controls shall be used . (the latter being normally 12–18 hr) selection is presented in the reference following treatment. Since the time under paragraph (g)(5) of this section). (3) Procedure—(i) Number and sex of required for uptake and metabolism of If there is toxicity, three dose levels animals. Each treated and control group the test substance as well as its effect on shall be used for the first sampling time. shall include at least 5 analyzable cell cycle kinetics can affect the These dose levels shall cover a range animals per sex. If at the time of the optimum time for chromosome from the maximum to little or no study there are data available from aberration detection, a later sample toxicity. At the later sampling time only studies in the same species and using collection 24 hr after the first sample the highest dose needs to be used. The the same route of exposure that time is recommended. If dose regimens highest dose is defined as the dose demonstrate that there are no of more than one day are used, one producing signs of toxicity such that substantial differences in toxicity sampling time at 1.5 normal cell cycle higher dose levels, based on the same between sexes, then testing in a single lengths after the final treatment should dosing regimen, would be expected to sex will be sufficient. Where human be used. produce lethality. Substances with exposure to chemicals may be sex- (C) Prior to sacrifice, animals shall be specific biological activities at low non- specific, as for example with some injected intraperitoneally with an toxic doses (such as hormones and pharmaceutical agents, the test should appropriate dose of a metaphase mitogens) may be exceptions to the 43852 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations dose-setting criteria and should be be presented in tabular form. The (vi) The likelihood that the test evaluated on a case-by-case basis. The experimental unit is the animal. For substance or its metabolites reach the highest dose may also be defined as a each animal the number of cells scored, general circulation or specifically the dose that produces some indication of the number of aberrations per cell and target tissue (e.g., systemic toxicity) toxicity in the bone marrow (e.g. greater the percentage of cells with structural should be discussed. than 50% reduction in mitotic index). chromosome aberration(s) shall be (3) Test report. The test report shall (iv) Limit test. If a test at one dose evaluated. Different types of structural include the following information: level of at least 2,000 mg/kg body chromosome aberrations shall be listed (i) Test substance: weight using a single treatment, or as with their numbers and frequencies for (A) Identification data and CAS No., two treatments on the same day, treated and control groups. Gaps shall if known. produces no observable toxic effects, be recorded separately and reported but (B) Physical nature and purity. and if genotoxicity would not be generally not included in the total (C) Physicochemical properties expected based on data from aberration frequency. If there is no relevant to the conduct of the study. structurally related compounds, then a evidence for a difference in response (D) Stability of the test substance, if full study using three dose levels may between the sexes, the data may be known. not be considered necessary. For studies combined for statistical analysis. (ii) Solvent/vehicle: of a longer duration, the limit dose is (2) Evaluation and interpretation of (A) Justification for choice of vehicle. 2,000 mg/kg/body weight/day for results. (i) There are several criteria for (B) Solubility and stability of the test treatment up to 14 days, and 1,000 mg/ determining a positive result, such as a substance in solvent/vehicle, if known. kg/body weight/day for treatment longer dose-related increase in the relative (iii) Test animals: than 14 days. Expected human exposure number of cells with chromosome (A) Species/strain used. may indicate the need for a higher dose aberrations or a clear increase in the (B) Number, age and sex of animals. level to be used in the limit test. number of cells with aberrations in a (C) Source, housing conditions, diet, (v) Administration of doses. The test single dose group at a single sampling etc. substance is usually administered by time. Biological relevance of the results (D) Individual weight of the animals gavage using a stomach tube or a should be considered first. Statistical at the start of the test, including body suitable intubation cannula, or by methods may be used as an aid in weight range, mean and standard intraperitoneal injection. Other routes of evaluating the test results (some deviation for each group. exposure may be acceptable where they statistical methods are described in the (iv) Test conditions: can be justified. The maximum volume reference under paragraph (g)(6) of this (A) Positive and negative (vehicle/ of liquid that can be administered by section). Statistical significance should solvent) controls. gavage or injection at one time depends (B) Data from range-finding study, if not be the only determining factor for a on the size of the test animal. The conducted. positive response. Equivocal results volume should not exceed 2 ml/100g (C) Rationale for dose level selection. should be clarified by further testing body weight. The use of volumes higher (D) Details of test substance preferably using a modification of than these must be justified. Except for preparation. experimental conditions. irritating or corrosive substances which (E) Details of the administration of the will normally reveal exacerbated effects (ii) An increase in polyploidy may test substance. with higher concentrations, variability indicate that the test substance has the (F) Rationale for route of in test volume should be minimized by potential to induce numerical administration. adjusting the concentration to ensure a chromosome aberrations. An increase in (G) Methods for verifying that the test constant volume at all dose levels. endoreduplication may indicate that the substance reached the general (vi) Chromosome preparation. test substance has the potential to circulation or target tissue, if applicable. Immediately after sacrifice, bone inhibit cell cycle progression. This (H) Conversion from diet/drinking marrow shall be obtained, exposed to phenomenon is described in the water test substance concentration parts hypotonic solution and fixed. The cells references under paragraphs (g)(7) and per million (ppm) to the actual dose shall be then spread on slides and (g)(8) of this section. (mg/kg body weight/day), if applicable. stained. (iii) A test substance for which the (I) Details of food and water quality. (vii) Analysis. (A) The mitotic index results do not meet the criteria (J) Detailed description of treatment should be determined as a measure of described in paragraph (f)(2)(i) of this and sampling schedules. cytotoxicity in at least 1,000 cells per section is considered non-mutagenic in (K) Methods for measurement of animal for all treated animals (including this test. toxicity. positive controls) and untreated (iv) Although most experiments will (L) Identity of metaphase arresting negative control animals. give clearly positive or negative results, substance, its concentration and (B) At least 100 cells should be in rare cases the data set will preclude duration of treatment. analyzed for each animal. This number making a definite judgment about the (M) Methods of slide preparation. could be reduced when high numbers of activity of the test substance. Results (N) Criteria for scoring aberrations. aberrations are observed. All slides, may remain equivocal or questionable (O) Number of cells analyzed per including those of positive and negative regardless of the number of experiments animal. controls, shall be independently coded performed. (P) Criteria for considering studies as before microscopic analysis. Since slide (v) Positive results from the in vivo positive, negative or equivocal. preparation procedures often result in chromosome aberration test indicate (v) Results: the breakage of a proportion of that a substance induces chromosome (A) Signs of toxicity. metaphases with loss of chromosomes, aberrations in the bone marrow of the (B) Mitotic index. the cells scored should therefore contain species tested. Negative results indicate (C) Type and number of aberrations, a number of centromeres equal to the that, under the test conditions, the test given separately for each animal. number 2n ± 2. substance does not induce chromosome (D) Total number of aberrations per (f) Data and reporting—(1) Treatment aberrations in the bone marrow of the group with means and standard of results. Individual animal data shall species tested. deviations. Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43853

(E) Number of cells with aberrations Ferguson, R., Richold, M., Papworth, chromosome fragments or whole per group with means and standard D.G., and Savage, J.R.K. Ed. Kirkland,D. chromosomes. deviations. J. Statistical Analysis of In Vivo Normochromatic erythrocyte is a (F) Changes in ploidy, if seen. Cytogenetic Assays. UKEMS Sub- mature erythrocyte that lacks ribosomes (G) Dose-response relationship, where Committee on Guidelines for and can be distinguished from possible. Mutagenicity Testing. Report Part III. immature, polychromatic erythrocytes (H) Statistical analyses, if any. Statistical Evaluation of Mutagenicity by stains selective for ribosomes. (I) Concurrent negative control data. Test Data (Cambridge University Press, Polychromatic erythrocyte is a (J) Historical negative control data Cambridge, 1989) pp. 184–232. immature erythrocyte, in an with ranges, means and standard (7) Locke-Huhle, C. intermediate stage of development, that deviations. Endoreduplication in Chinese Hamster still contains ribosomes and therefore (K) Concurrent positive control data. Cells During Alpha-Radiation Induced can be distinguished from mature, (vi) Discussion of the results. G2 Arrest. Mutation Research. 119, 403– normochromatic erythrocytes by stains (vii) Conclusion. 413 (1983). selective for ribosomes. (g) References. For additional (8) Huang, Y., Change, C., and Trosko, (d) Initial considerations. (1) The bone background information on this test J. E. Aphidicolin-Induced marrow of rodents is routinely used in guideline, the following references Endoreduplication in Chinese Hamster this test since polychromatic should be consulted. These references Cells. Cancer Research. 43, 1362–1364 erythrocytes are produced in that tissue. are available for inspection at the TSCA (1983). The measurement of micronucleated Nonconfidential Information Center, immature (polychromatic) erythrocytes Rm. NE–B607, Environmental § 799.9539 TSCA mammalian erythrocyte in peripheral blood is equally Protection Agency, 401 M St., SW., micronucleus test. acceptable in any species in which the Washington, DC, 12 noon to 4 p.m., (a) Scope. This section is intended to inability of the spleen to remove Monday through Friday, except legal meet the testing requirements under micronucleated erythrocytes has been holidays. section 4 of TSCA. demonstrated, or which has shown an (1) Adler, I.D. Eds. S. Venitt and J.M. (1) The mammalian erythrocyte adequate sensitivity to detect agents that Parry. Cytogenetic Tests in Mammals. micronucleus test is used for the cause structural or numerical Mutagenicity Testing: A Practical detection of damage induced by the test chromosome aberrations. Micronuclei Approach. (IRL Press, Oxford, substance to the chromosomes or the can be distinguished by a number of Washington DC, 1984) pp. 275–306. mitotic apparatus of erythroblasts by criteria. These include identification of (2) Preston, R.J., Dean, B.J., Galloway, analysis of erythrocytes as sampled in the presence or absence of a kinetochore S., Holden, H., McFee, A.F., and Shelby, bone marrow and/or peripheral blood or centromeric DNA in the micronuclei. M. Mammalian In Vivo Cytogenetic cells of animals, usually rodents. The frequency of micronucleated Assays: Analysis of Chromosome (2) The purpose of the micronucleus immature (polychromatic) erythrocytes Aberrations in Bone Marrow Cells. test is to identify substances that cause is the principal endpoint. The number Mutation Research. 189, 157–165 cytogenetic damage which results in the of mature (normochromatic) (1987). formation of micronuclei containing erythrocytes in the peripheral blood that (3) Richold, M., Chandley, A., Ashby, lagging chromosome fragments or whole contain micronuclei among a given J., Gatehouse, D.G., Bootman, J., and chromosomes. number of mature erythrocytes can also Henderson, L. Ed. D.J. Kirkland. In Vivo (3) When a bone marrow erythroblast be used as the endpoint of the assay Cytogenetic Assays. Basic Mutagenicity develops into a polychromatic when animals are treated continuously Tests, UKEMS Recommended erythrocyte, the main nucleus is for 4 weeks or more. This mammalian Procedures. UKEMS Subcommittee on extruded; any micronucleus that has in vivo micronucleus test is especially Guidelines for Mutagenicity Testing. been formed may remain behind in the relevant to assessing mutagenic hazard Report. Part I revised. (Cambridge otherwise anucleated cytoplasm. in that it allows consideration of factors University Press, Cambridge, NY, Port Visualization of micronuclei is of in vivo metabolism, pharmacokinetics Chester, Melbourne, Sydney, 1990) pp. facilitated in these cells because they and DNA-repair processes although 115–141. lack a main nucleus. An increase in the these may vary among species, among (4) Tice, R.R., Hayashi, M., frequency of micronucleated tissues and among genetic endpoints. MacGregor, J.T., Anderson, D., Blakey, polychromatic erythrocytes in treated An in vivo assay is also useful for D.H., Holden, H.E., Kirsch-Volders, M., animals is an indication of induced further investigation of a mutagenic Oleson Jr., F.B., Pacchierotti, F., Preston, chromosome damage. effect detected by an in vitro system. R.J., Romagna, F., Shimada, H., Sutou, (b) Source. The source material used (2) If there is evidence that the test S., and Vannier, B. Report from the in developing this TSCA test guideline substance, or a reactive metabolite, will Working Group on the In Vivo is the OECD guideline 474 (February not reach the target tissue, it is not Mammalian Bone Marrow Chromosomal 1997). This source is available at the appropriate to use this test. Aberration Test. Mutation Research. address in paragraph (g) of this section. (e) Test method—(1) Principle. 312, 305–312 (1994). (c) Definitions. The following Animals are exposed to the test (5) Fielder, R.J., Allen, J.A., Boobis, definitions apply to this section: substance by an appropriate route. If A.R., Botham, P.A., Doe, J., Esdaile, D.J., Centromere (kinetochore) is a region bone marrow is used, the animals are Gatehouse, D.G., Hodson-Walker, G., of a chromosome with which spindle sacrificed at appropriate times after Morton, D.B., Kirkland, D. J., and fibers are associated during cell treatment, the bone marrow extracted, Richold, M. Report of British Toxicology division, allowing orderly movement of and preparations made and stained (test Society/UK Environmental Mutagen daughter chromosomes to the poles of techniques described in the references Society Working Group: Dose Setting in the daughter cells. under paragraphs (g)(1), (g)(2), and (g)(3) In Vivo Mutagenicity Assays. Micronuclei are small nuclei, separate of this section may be used). When Mutagenesis. 7, 313–319 (1992). from and additional to the main nuclei peripheral blood is used, the blood is (6) Lovell, D.P., Anderson, D., of cells, produced during telophase of collected at appropriate times after Albanese, R., Amphlett, G.E., Clare, G., mitosis (meiosis) by lagging treatment and smear preparations are 43854 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations made and stained (the test techniques exceed 70% other than during room produce toxic effects at the dose levels described in the references under cleaning, the aim should be 50–60%. used, and should not be suspected of paragraphs (g)(3), (g)(4), (g)(5), and (g)(6) Lighting should be artificial, the chemical reaction with the test of this section may be used). For studies sequence being 12 hrs light, 12 hrs dark. substance. If other than well-known with peripheral blood, as little time as For feeding, conventional laboratory solvents/vehicles are used, their possible should elapse between the last diets may be used with an unlimited inclusion shall be supported with exposure and cell harvest. Preparations supply of drinking water. The choice of reference data indicating their are analyzed for the presence of diet may be influenced by the need to compatibility. It is recommended that micronuclei. ensure a suitable admixture of a test wherever possible, the use of an (2) Description—(i) Preparations—(A) substance when administered by this aqueous solvent/vehicle should be Selection of animal species. Mice or rats route. Animals may be housed considered first. are recommended if bone marrow is individually, or caged in small groups of (B) Controls. (1) Concurrent positive used, although any appropriate the same sex. and negative (solvent/vehicle) controls mammalian species may be used. When (C) Preparation of the animals. shall be included for each sex in each peripheral blood is used, mice are Healthy young adult animals shall be test. Except for treatment with the test randomly assigned to the control and recommended. However, any substance, animals in the control groups treatment groups. The animals are appropriate mammalian species may be should be handled in an identical identified uniquely. The animals are used provided it is a species in which manner to animals of the treatment acclimated to the laboratory conditions the spleen does not remove groups. for at least 5 days. Cages should be micronucleated erythrocytes or a arranged in such a way that possible (2) Positive controls shall produce species which has shown an adequate effects due to cage placement are micronuclei in vivo at exposure levels sensitivity to detect agents that cause minimized. expected to give a detectable increase structural or numerical chromosome (D) Preparation of doses. Solid test over background. Positive control doses aberrations. Commonly used laboratory substances shall be dissolved or should be chosen so that the effects are strains of young healthy animals should suspended in appropriate solvents or clear but do not immediately reveal the be employed. At the commencement of vehicles and diluted, if appropriate, identity of the coded slides to the the study, the weight variation of prior to dosing of the animals. Liquid reader. It is acceptable that the positive animals should be minimal and not control be administered by a route ± test substances may be dosed directly or exceed 20% of the mean weight of diluted prior to dosing. Fresh different from the test substance and each sex. preparations of the test substance sampled at only a single time. In (B) Housing and feeding conditions. should be employed unless stability addition, the use of chemical class- The temperature in the experimental data demonstrate the acceptability of related positive control chemicals may animal room should be 22°C (± 3°C). storage. be considered, when available. Although the relative humidity should (ii) Test conditions—(A) Solvent/ Examples of positive control substances be at least 30% and preferably not vehicle. The solvent/vehicle should not include:

Chemical CAS No.

Ethyl methanesulphonate ...... [CAS no. 62±50±0] Ethyl nitrosourea ...... [CAS no. 759±73±9] Mitomycin C ...... [CAS no. 50±07±7] Cyclophosphamide (monohydrate) ...... [CAS no. 50±18±0] [CAS no. 6055±19±2] Triethylenemelamine ...... [CAS no. 51±18±3]

(3) Negative controls, treated with blood studies (e.g., one to three agents, the test should be performed solvent or vehicle alone, and otherwise treatment(s)) when the resulting data are with animals of the appropriate sex. treated in the same way as the treatment in the expected range for the historical (ii) Treatment schedule. (A) No groups shall be included for every control. standard treatment schedule (i.e. one, sampling time, unless acceptable inter- (3) Procedure—(i) Number and sex of two, or more treatments at 24 h animal variability and frequencies of animals. Each treated and control group intervals) can be recommended. The cells with micronuclei are demonstrated shall include at least 5 analyzable samples from extended dose regimens by historical control data. If single animals per sex (techniques described are acceptable as long as a positive sampling is applied for negative in the reference under paragraph (g)(7) effect has been demonstrated for this controls, the most appropriate time is of this section may be used). If at the study or, for a negative study, as long as the first sampling time. In addition, untreated controls should also be used time of the study there are data available toxicity has been demonstrated or the unless there are historical or published from studies in the same species and limit dose has been used, and dosing control data demonstrating that no using the same route of exposure that continued until the time of sampling. deleterious or mutagenic effects are demonstrate that there are no Test substances may also be induced by the chosen solvent/vehicle. substantial differences between sexes in administered as a split dose, i.e., two toxicity, then testing in a single sex will treatments on the same day separated by (4) If peripheral blood is used, a pre- be sufficient. Where human exposure to no more than a few hrs, to facilitate treatment sample may also be chemicals may be sex-specific, as for administering a large volume of acceptable as a concurrent negative example with some pharmaceutical material. control, but only in the short peripheral Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43855

(B) The test may be performed in two structurally related substances, then a section maybe used). All slides, ways: full study using three dose levels may including those of positive and negative (1) Animals shall be treated with the not be considered necessary. For studies controls, shall be independently coded test substance once. Samples of bone of a longer duration, the limit dose is before microscopic analysis. At least marrow shall be taken at least twice, 2,000 mg/kg/body weight/day for 2,000 immature erythrocytes per animal starting not earlier than 24 hrs after treatment up to 14 days, and 1,000 mg/ shall be scored for the incidence of treatment, but not extending beyond 48 kg/body weight/day for treatment longer micronucleated immature erythrocytes. hrs after treatment with appropriate than 14 days. Expected human exposure Additional information may be obtained interval(s) between samples. The use of may indicate the need for a higher dose by scoring mature erythrocytes for sampling times earlier than 24 hrs after level to be used in the limit test. micronuclei. When analyzing slides, the treatment should be justified. Samples (v) Administration of doses. The test proportion of immature erythrocytes of peripheral blood shall be taken at substance is usually administered by among total erythrocytes should not be least twice, starting not earlier than 36 gavage using a stomach tube or a less than 20% of the control value. hrs after treatment, with appropriate suitable intubation cannula, or by When animals are treated continuously intervals following the first sample, but intraperitoneal injection. Other routes of for 4 weeks or more, at least 2,000 not extending beyond 72 hrs. When a exposure may be acceptable where they mature erythrocytes per animal can also positive response is recognized at one can be justified. The maximum volume be scored for the incidence of sampling time, additional sampling is of liquid that can be administered by micronuclei. Systems for automated not required. gavage or injection at one time depends analysis (image analysis) and cell (2) If two or more daily treatments are on the size of the test animal. The suspensions (flow cytometry) are used (e.g. two or more treatments at 24 volume should not exceed 2 ml/100g acceptable alternatives to manual hr intervals), samples shall be collected body weight. The use of volumes higher evaluation if appropriately justified and once between 18 and 24 hrs following than these must be justified. Except for validated. the final treatment for the bone marrow irritating or corrosive substances which (f) Data and reporting—(1) Treatment and once between 36 and 48 hrs will normally reveal exacerbated effects of results. Individual animal data shall following the final treatment for the with higher concentrations, variability be presented in tabular form. The peripheral blood (techniques described in test volume should be minimized by experimental unit is the animal. The in the reference under paragraph (g)(8) adjusting the concentration to ensure a number of immature erythrocytes of this section may be used). constant volume at all dose levels. scored, the number of micronucleated (C) Other sampling times may be used (vi) Bone marrow/blood preparation. immature erythrocytes, and the number in addition, when relevant. Bone marrow cells shall be obtained of immature among total erythrocytes (iii) Dose levels. If a range finding from the femurs or tibias immediately shall be listed separately for each study is performed because there are no following sacrifice. Cells shall be animal analyzed. When animals are suitable data available, it should be removed from femurs or tibias, prepared treated continuously for 4 weeks or performed in the same laboratory, using and stained using established methods. more, the data on mature erythrocytes the same species, strain, sex, and Peripheral blood is obtained from the should also be given if it is collected. treatment regimen to be used in the tail vein or other appropriate blood The proportion of immature among total main study (guidance on dose setting is vessel. Blood cells are immediately erythrocytes and, if considered provided in the reference in paragraph stained supravitally (the test techniques applicable, the percentage of (g)(9) of this section). If there is toxicity, described in the references under micronucleated erythrocytes shall be three dose levels shall be used for the paragraphs (g)(4), (g)(5), and (g)(6) of given for each animal. If there is no first sampling time. These dose levels this section may be used) or smear evidence for a difference in response shall cover a range from the maximum preparations are made and then stained. between the sexes, the data from both to little or no toxicity. At the later The use of a DNA specific stain (e.g. sexes may be combined for statistical sampling time only the highest dose acridine orange (techniques described in analysis. needs to be used. The highest dose is the reference under paragraph (g)(10) of (2) Evaluation and interpretation of defined as the dose producing signs of this section may be used) or Hoechst results. (i) There are several criteria for toxicity such that higher dose levels, 33258 plus pyronin-Y) can eliminate determining a positive result, such as a based on the same dosing regimen, some of the artifacts associated with dose-related increase in the number of would be expected to produce lethality. using a non-DNA specific stain. This micronucleated cells or a clear increase Substances with specific biological advantage does not preclude the use of in the number of micronucleated cells activities at low non-toxic doses (such conventional stains (e.g., Giemsa). in a single dose group at a single as hormones and mitogens) may be Additional systems (e.g. cellulose sampling time. Biological relevance of exceptions to the dose-setting criteria columns to remove nucleated cells (the the results should be considered first. and should be evaluated on a case-by- test techniques described in the Statistical methods may be used as an case basis. The highest dose may also be references under paragraph (g)(12) of aid in evaluating the test results (the test defined as a dose that produces some this section may be used)) can also be techniques described in the references indication of toxicity in the bone used provided that these systems have paragraphs (g)(14) and (g)(15) of this marrow (e.g. a reduction in the been shown to adequately work for section may be used). Statistical proportion of immature erythrocytes micronucleus preparation in the significance should not be the only among total erythrocytes in the bone laboratory. determining factor for a positive marrow or peripheral blood). (vii) Analysis. The proportion of response. Equivocal results should be (iv) Limit test. If a test at one dose immature among total (immature + clarified by further testing preferably level of at least 2,000 mg/kg body mature) erythrocytes is determined for using a modification of experimental weight using a single treatment, or as each animal by counting a total of at conditions. two treatments on the same day, least 200 erythrocytes for bone marrow (ii) A test substance for which the produces no observable toxic effects, and 1,000 erythrocytes for peripheral results do not meet the criteria and if genotoxicity would not be blood (techniques described in the described is considered non-mutagenic expected based upon data from reference under paragraph (g)(13) of this in this test. 43856 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(iii) Although most experiments will (I) Details of food and water quality. Collaborative Study by CSGMT/JEMS. give clearly positive or negative results, (J) Detailed description of treatment MMS. Mutation Research. 278, 83–98. in rare cases the data set will preclude and sampling schedules. (6) The Collaborative Study Group for making a definite judgement about the (K) Methods of slide preparation. the Micronucleus Test (CSGMT/ activity of the test substance. Results, (L) Methods for measurement of JEMMS.MMS, The Mammalian may remain equivocal or questionable toxicity. Mutagenesis Study Group of the regardless of the number of times the (M) Criteria for scoring Environmental Mutagen Society of experiment is repeated. Positive results micronucleated immature erythrocytes. Japan) Protocol recommended for the in the micronucleus test indicate that a (N) Number of cells analyzed per short-term mouse peripheral blood substance induces micronuclei which animal. micronucleus test. Mutagenesis. 10, are the result of chromosomal damage or (O) Criteria for considering studies as 153–159 (1995). damage to the mitotic apparatus in the positive, negative or equivocal. (7) Hayashi, M., Tice, R.R., erythroblasts of the test species. (v) Results: MacGregor, J.T., Anderson, D., Blakey, Negative results indicate that, under the (A) Signs of toxicity. D.H., Kirsch-Volders, M., Oleson, Jr. test conditions, the test substance does (B) Proportion of immature F.B., Pacchierotti, F., Romagna, F., not produce micronuclei in the erythrocytes among total erythrocytes. Shimada, H., Sutou, S., and Vannier, B. immature erythrocytes of the test (C) Number of micronucleated In Vivo Rodent Erythrocyte species. immature erythrocytes, given separately Micronucleus Assay. Mutation for each animal. Research. 312, 293–304 (1994). (iv) The likelihood that the test ± substance or its metabolites reach the (D) Mean standard deviation of (8) Higashikuni, N. and Sutou, S. An general circulation or specifically the micronucleated immature erythrocytes optimal, generalized sampling time of target tissue (e.g. systemic toxicity) per group. 30 ∂/- 6 h after double dosing in the should be discussed. (E) Dose-response relationship, where mouse peripheral blood micronucleus (3) Test report. In addition to the possible. test. Mutagenesis. 10, 313–319 (1995). reporting requirements as specified (F) Statistical analyses and method (9) Fielder, R.J., Allen, J.A., Boobis, under 40 CFR part 792, subpart J, the applied. A.R., Botham, P.A., Doe, J., Esdaile, D.J., following specific information shall be (G) Concurrent and historical negative Gatehouse, D.G., Hodson-Walker, G., reported. Both individual and summary control data. Morton, D.B., Kirkland, D. J., and data should be presented. (H) Concurrent positive control data. Richold, M. Report of British Toxicology (i) Test substance: (vi) Discussion of the results. Society/UK Environmental Mutagen (A) Identification data and CAS no., if (vii) Conclusion. Society Working Group: Dose Setting in known. (g) References. For additional In Vivo Mutagenicity Assays. (B) Physical nature and purity. background information on this test Mutagenesis. 7, 313–319 (1992). (C) Physiochemical properties guideline, the following references (10) Hayashi, M., Sofuni, T., and relevant to the conduct of the study. should be consulted. These references Ishidate, Jr., M. An Application of (D) Stability of the test substance, if are available for inspection at the TSCA Acridine Orange Fluorescent Staining to known. Nonconfidential Information Center, the Micronucleus Test. Mutation (ii) Solvent/vehicle: Rm. NE–B607, Environmental Research. 120, 241–247 (1983). (A) Justification for choice of vehicle. Protection Agency, 401 M St., SW., (11) MacGregor, J.T., Wehr, C.M., and (B) Solubility and stability of the test Washington, DC, 12 noon to 4 p.m., Langlois, R.G. A Simple Fluorescent substance in the solvent/vehicle, if Monday through Friday, except legal Staining Procedure for Micronuclei and known. holidays. RNA in Erythrocytes Using Hoechst (iii) Test animals: (1) Heddle, J.A. A Rapid In Vivo Test 33258 and Pyronin Y. Mutation (A) Species/strain used. for Chromosomal Damage. Mutation (B) Number, age, and sex of animals. Research. 120, 269–275 (1983). Research. 18, 187–190 (1973). (12) Romagna, F. and Staniforth, C.D. (C) Source, housing conditions, diet, (2) Schmid, W. The Micronucleus The automated bone marrow etc. Test. Mutation Research. 31, 9–15 (D) Individual weight of the animals micronucleus test. Mutation Research. (1975). at the start of the test, including body 213, 91–104 (1989). (3) Mavournin, K.H., Blakey, D.H., (13) Gollapudi, B. and McFadden, weight range, mean and standard Cimino, M.C., Salamone, M.F., and L.G. Sample size for the estimation of deviation for each group. Heddle, J.A. The In Vivo Micronucleus (iv) Test conditions: polychromatic to normochromatic (A) Positive and negative (vehicle/ Assay in Mammalian Bone Marrow and eruthrocyte ratio in the bone marrow solvent) control data. Peripheral Blood. A report of the U.S. micronucleus test. Mutation Research. (B) Data from range-finding study, if Environmental Protection Agency Gene- 347, 97–99 (1995). conducted. Tox Program. Mutation Research. 239, (14) Richold, M., Ashby, J., Bootman, (C) Rationale for dose level selection. 29–80 (1990). J., Chandley, A., Gatehouse, D.G., and (D) Details of test substance (4) Hayashi, M., Morita, T., Kodama, Henderson, L. Ed. Kirkland, D.J. In Vivo preparation. Y., Sofuni, T., and Ishidate, Jr., M. The Cytogenetics Assays. Basic Mutagenicity (E) Details of the administration of the Micronucleus Assay with Mouse Tests, UKEMS Recommended test substance. Peripheral Blood Reticulocytes Using Procedures. UKEMS Subcommittee on (F) Rationale for route of Acridine Orange–Coated Slides. Guidelines for Mutagenicity Testing. administration. Mutation Research. 245, 245–249 Report. Part I revised (Cambridge (G) Methods for verifying that the test (1990). University Press, Cambridge, New York, substance reached the general (5) The Collaborative Study Group for Port Chester, Melbourne, Sydney, 1990) circulation or target tissue, if applicable. the Micronucleus Test (1992). pp. 115–141. (H) Conversion from diet/drinking Micronucleus Test with Mouse (15) Lovell, D.P., Anderson, D., water test substance concentration parts Peripheral Blood Erythrocytes by Albanese, R., Amphlett, G.E., Clare, G., per million (ppm) to the actual dose Acridine Orange Supravital Staining: Ferguson, R., Richold, M., Papworth, (mg/kg body weight/day), if applicable. The Summary Report of the 5th D.G., and Savage, J.R.K. Ed. D.J. Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43857

Kirkland. Statistical Analysis of In Vivo battery is not intended to provide a scheduled to be perfused before the end Cytogenetic Assays. Statistical complete evaluation of neurotoxicity, of the study. Animals shall be randomly Evaluation of Mutagenicity Test Data. and additional functional and assigned to treatment and control UKEMS Sub-Committee on Guidelines morphological evaluation may be groups. for Mutagenicity Testing, Report, Part necessary to assess completely the (3) Control groups. (i) A concurrent III. (Cambridge University Press, neurotoxic potential of a chemical. (vehicle) control group is required. Cambridge, New York, Port Chester, (b) Source. The source material used Subjects shall be treated in the same Melbourne, Sydney, 1989) pp. 184–232. in developing this TSCA test guideline way as for an exposure group except (16) Heddle, J.A., Salamone, M.F., is the OPPTS harmonized test guideline that administration of the test substance Hite, M., Kirkhart, B., Mavournin, K., 870.6200 (June 1996 Public Draft). This is omitted. If the vehicle used has MacGregor, J.G., and Newell, G.W. The source is available at the address in known or potential toxic properties, Induction of Micronuclei as a Measure paragraph (g) of this section. both untreated or saline treated and of Genotoxicity. Mutation Research. (c) Definitions. The following vehicle control groups are required. 123: 61–118 (1983). definitions apply to this section. (ii) Positive control data from the (17) MacGregor, J.T., Heddle, J.A., ED is effective dose. laboratory performing the testing shall Hite, M., Margolin, G.H., Ramel C., Motor activity is any movement of the provide evidence of the ability of the Salamone, M.F., Tice, R.R., and Wild, D. experimental animal. observational methods used to detect Guidelines for the Conduct of Neurotoxicity is any adverse effect on major neurotoxic endpoints including Micronucleus Assays in Mammalian the structure or function of the nervous limb weakness or paralysis, tremor, and Bone Marrow Erythrocytes. Mutation system related to exposure to a chemical autonomic signs. Positive control data Research. 189: 103–112 (1987). substance. are also required to demonstrate the (18) MacGregor, J.T., Wehr, C.M., Toxic effect is an adverse change in sensitivity and reliability of the activity- Henika, P.R., and Shelby, M.E. (1990). the structure or function of an measuring device and testing The In Vivo Erythrocyte Micronucleus experimental animal as a result of procedures. These data should Test: Measurement at Steady State exposure to a chemical substance. demonstrate the ability to detect Increases Assay Efficiency and Permits (d) Principle of the test method. The chemically induced increases and Integration with Toxicity Studies. test substance is administered to several decreases in activity. Positive control Fundamental Applied Toxicology. 14: groups of experimental animals, one groups exhibiting central nervous 513–522. dose being used per group. The animals system pathology and peripheral (19) MacGregor, J.T., Schlegel, R. are observed under carefully nervous system pathology are also Choy, W.N., and Wehr, C.M. Eds. Hayes, standardized conditions with sufficient required. Separate groups for peripheral A.W., Schnell, R.C., and Miya, T.S. frequency to ensure the detection and and central neuropathology are Micronuclei in Circulating Erythrocytes: quantification of behavioral and/or acceptable (e.g. acrylamide and A Rapid Screen for Chromosomal neurologic abnormalities, if present. trimethyl tin). Positive control data shall Damage During Routine Toxicity Various functions that could be affected be collected at the time of the test study Testing in Mice. Developments in by neurotoxicants are assessed during unless the laboratory can demonstrate Science and Practice of Toxicology each observation period. Measurements the adequacy of historical data for this (Elsevier, Amsterdam, 1983) pp. 555– of motor activity of individual animals purpose, i.e. by the approach outlined 558. are made in an automated device. The in this section. animals are perfused and tissue samples (4) Dose level and dose selection. At § 799.9620 TSCA neurotoxicity screening from the nervous system are prepared least three doses shall be used in battery. for microscopic examination. The addition to the vehicle control group. (a) Scope. This section is intended to exposure levels at which significant The data should be sufficient to produce meet the testing requirements under neurotoxic effects are produced are a dose-effect curve. The Agency strongly section 4 of TSCA. This neurotoxicity compared to one another and to those encourage the use of equally spaced screening battery consists of a levels that produce other toxic effects. doses and a rationale for dose selection functional observational battery, motor (e) Test procedures—(1) Animal that will maximally support detection of activity, and neuropathology. The selection—(i) Species. In general, the dose-effect relations. For acute studies, functional observational battery consists laboratory rat should be used. Under dose selection may be made relative to of noninvasive procedures designed to some circumstances, other species, such the establishment of a benchmark dose detect gross functional deficits in as the mouse or the dog, may be more (BD). That is, doses may be specified as animals and to better quantify appropriate, although not all of the successive fractions, e.g. 0.5, 0.25, ...n of behavioral or neurological effects battery may be adaptable to other the BD. The BD itself may be estimated detected in other studies. The motor species. as the highest nonlethal dose as activity test uses an automated device (ii) Age. Young adults (at least 42 days determined in a preliminary range- that measures the level of activity of an old for rats) shall be used. finding lethality study. A variety of test individual animal. The (iii) Sex. Both males and females shall methodologies may be used for this neuropathological techniques are be used. Females shall be nulliparous purpose, and the method chosen may designed to provide data to detect and and nonpregnant. influence subsequent dose selection. characterize histopathological changes (2) Number of animals. At least 10 The goal is to use a dose level that is in the central and peripheral nervous males and 10 females should be used in sufficient to be judged a limit dose, or system. This battery is designed to be each dose and control group for clearly toxic. used in conjunction with general behavioral testing. At least five males (i) Acute studies. The high dose need toxicity studies and changes should be and five females should be used in each not be greater than 2 g/kg. Otherwise, evaluated in the context of both the dose and control group for terminal the high dose should result in concordance between functional neuropathology. If interim significant neurotoxic effects or other neurological and neuropatholgical neuropathological evaluations are clearly toxic effects, but not result in an effects, and with respect to any other planned, the number should be incidence of fatalities that would toxicological effects seen. This test increased by the number of animals preclude a meaningful evaluation of the 43858 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations data. This dose may be estimated by a (ii) Functional observational battery— the cage or handling, with a range of BD procedure as described under (A) General conduct. All animals in a severity scores from no reaction to paragraph (e)(4) of this section, with the given study shall be observed carefully hyperreactivity. middle and low dose levels chosen as by trained observers who are unaware of (4) Ranking of the subject’s general fractions of the BD dose. The lowest the animals’ treatment, using level of activity during observations of dose should produce minimal effect, e.g. standardized procedures to minimize the unperturbed subject in the open an ED10, or alternatively, no effects. observer variability. Where possible, it field, with a range of severity scores (ii) Subchronic and chronic studies. is advisable that the same observer be from unresponsive to hyperactive. The high dose need not be greater than used to evaluate the animals in a given (5) Descriptions and incidence of 1 g/kg. Otherwise, the high dose level study. If this is not possible, some posture and gait abnormalities observed should result in significant neurotoxic demonstration of interobserver in the home cage and open field. effects or other clearly toxic effects, but reliability is required. The animals shall (6) Ranking of any gait abnormalities, not produce an incidence of fatalities be removed from the home cage to a with a range of severity scores from that would prevent a meaningful standard arena for observation. Effort none to severe. evaluation of the data. The middle and should be made to ensure that variations (7) Forelimb and hindlimb grip low doses should be fractions of the in the test conditions are minimal and strength measured using an objective high dose. The lowest dose should are not systematically related to procedure (the procedure described in produce minimal effects, e.g. an ED10, treatment. Among the variables that can the reference under paragraph (g)(8) of or alternatively, no effects. affect behavior are sound level, this section may be used). (5) Route of exposure. Selection of temperature, humidity, lighting, odors, (8) Quantitative measure of landing route may be based on several criteria time of day, and environmental foot splay (the procedure described in including, the most likely route of distractions. Explicit, operationally the reference under paragraph (g)(3) of human exposure, bioavailability, the defined scales for each measure of the this section may be used). likelihood of observing effects, practical battery are to be used. The development (9) Sensorimotor responses to stimuli difficulties, and the likelihood of of objective quantitative measures of the of different modalities will be used to producing nonspecific effects. For many observational end-points specified is detect gross sensory deficits. Pain materials, it should be recognized that encouraged. Examples of observational perception may be assessed by a ranking more than one route of exposure may be procedures using defined protocols may or measure of the reaction to a tail- important and that these criteria may be found in the references under pinch, tail-flick, or hot-plate. The conflict with one another. Initially only paragraphs (g)(5), (g)(6), and (g)(9) of response to a sudden sound, e.g., click one route is required for screening for this section. The functional or snap, may be used to assess audition. neurotoxicity. The route that best meets observational battery shall include a (10) Body weight. (11) Description and incidence of any these criteria should be selected. Dietary thorough description of the subject’s unusual or abnormal behaviors, feeding will generally be acceptable for appearance, behavior, and functional excessive or repetitive actions repeated exposures studies. integrity. This shall be assessed through (stereotypies), emaciation, dehydration, (6) Combined protocol. The tests observations in the home cage and hypotonia or hypertonia, altered fur described in this screening battery may while the rat is moving freely in an open appearance, red or crusty deposits be combined with any other toxicity field, and through manipulative tests. around the eyes, nose, or mouth, and study, as long as none of the Testing should proceed from the least to any other observations that may requirements of either are violated by the most interactive with the subject. facilitate interpretation of the data. the combination. Scoring criteria, or explicitly defined scales, should be developed for those (C) Additional measures. Other (7) Study conduct—(i) Time of testing. measures may also be included and the All animals shall be weighed on each measures which involve subjective ranking. development and validation of new tests test day and at least weekly during the is encouraged. Further information on exposure period. (B) List of measures. The functional observational battery shall include the the neurobehavioral integrity of the (A) Acute studies. At a minimum, for subject may be provided by: acute studies observations and activity following list of measures: (1) Assessment of signs of autonomic (1) Count of rearing activity on the testing shall be made before the open field. initiation of exposure, at the estimated function, including but not limited to: (i) Ranking of the degree of (2) Ranking of righting ability. time of peak effect within 8 hrs of lacrimation and salivation, with a range (3) Body temperature. dosing, and at 7 and 14 days after of severity scores from none to severe. (4) Excessive or spontaneous dosing. Estimation of times of peak (ii) Presence or absence of vocalizations. effect may be made by dosing pairs of piloerection and exophthalmus. (5) Alterations in rate and ease of rats across a range of doses and making (iii) Ranking or count of urination and respiration, e.g., rales or dyspnea. regular observations of gait and arousal. defecation, including polyuria and (6) Sensorimotor responses to visual (B) Subchronic and chronic studies. diarrhea. This is most easily conducted or proprioceptive stimuli. In a subchronic study, at a minimum, during the open field assessment. (iii) Motor activity. Motor activity observations and activity measurements (iv) Pupillary function such as shall be monitored by an automated shall be made before the initiation of constriction of the pupil in response to activity recording apparatus. The device exposure and before the daily exposure, light or a measure of pupil size. used must be capable of detecting both or for feeding studies at the same time (v) Degree of palpebral closure, e.g., increases and decreases in activity, i.e., of day, during the 4th, 8th, and 13th ptosis. baseline activity as measured by the weeks of exposure. In chronic studies, at (2) Description, incidence, and device must not be so low as to preclude a minimum, observations and activity severity of any convulsions, tremors, or detection of decreases nor so high as to measurements shall be made before the abnormal motor movements, both in the preclude detection of increases in initiation of exposure and before the home cage and the open field. activity. Each device shall be tested by daily exposure, or for feeding studies at (3) Ranking of the subject’s reactivity standard procedures to ensure, to the the same time of day, every 3 months. to general stimuli such as removal from extent possible, reliability of operation Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43859 across devices and across days for any section may be used). Tissue blocks and ranging from very slight to very one device. In addition, treatment slides should be appropriately extensive. groups must be balanced across devices. identified when stored. Histological (f) Data reporting and evaluation. The Each animal shall be tested sections should be stained for final test report shall include the individually. The test session shall be hematoxylin and eosin (H&E), or a following information: long enough for motor activity to comparable stain according to standard (1) Description of equipment and test approach asymptotic levels by the last published protocols (some of these methods. A description of the general 20% of the session for nontreated protocols are described in the references design of the experiment and any control animals. All sessions shall have under paragraphs (g)(1) and (g)(11) of equipment used shall be provided. This the same duration. Treatment groups this section). shall include a short justification shall be counterbalanced across test (B) Qualitative examination. explaining any decisions involving times. Effort should be made to ensure Representative histological sections professional judgment. that variations in the test conditions are from the tissue samples should be (i) A detailed description of the minimal and are not systematically examined microscopically by an procedures used to standardize related to treatment. Among the appropriately trained pathologist for observations, including the arena and variables which can affect motor activity evidence of neuropathological scoring criteria. are sound level, size and shape of the alterations. The nervous system shall be (ii) Positive control data from the test cage, temperature, relative thoroughly examined for evidence of laboratory performing the test that humidity, lighting conditions, odors, any treatment-related neuropathological demonstrate the sensitivity of the use of the home cage or a novel test alterations. Particular attention should procedures being used. Historical data cage, and environmental distractions. be paid to regions known to be sensitive may be used if all essential aspects of (iv) Neuropathology: Collection, to neurotoxic insult or those regions the experimental protocol are the same. processing and examination of tissue likely to be affected based on the results Historical control data can be critical in samples. To provide for adequate of functional tests. Such treatment- the interpretation of study findings. The sampling as well as optimal related neuropathological alterations Agency encourages submission of such preservation of cellular integrity for the should be clearly distinguished from data to facilitate the rapid and complete detection of neuropathological artifacts resulting from influences other review of the significance of effects alterations, tissue shall be prepared for than exposure to the test substance. A seen. histological analysis using in situ stepwise examination of tissue samples (2) Results. The following information perfusion and paraffin and/or plastic is recommended. In such a stepwise shall be arranged by test group dose embedding procedures. Paraffin examination, sections from the high level. embedding is acceptable for tissue dose group are first compared with (i) In tabular form, data for each samples from the central nervous those of the control group. If no animal shall be provided showing: system. Plastic embedding of tissue neuropathological alterations are (A) Its identification number. samples from the central nervous observed in samples from the high dose (B) Its body weight and score on each system is encouraged, when feasible. group, subsequent analysis is not sign at each observation time, the time Plastic embedding is required for tissue required. If neuropathological and cause of death (if appropriate), total samples from the peripheral nervous alterations are observed in samples from session activity counts, and intrasession system. Subject to professional the high dose group, samples from the subtotals for each day measured. judgment and the type of intermediate and low dose groups are (ii) Summary data for each group neuropathological alterations observed, then examined sequentially. must include: it is recommended that additional (C) Subjective diagnosis. If any (A) The number of animals at the start methods, such as glial fibrillary acidic evidence of neuropathological of the test. protein (GFAP) immunohistochemistry alterations is found in the qualitative (B) The number of animals showing and/or methods known as Bodian’s or examination, then a subjective diagnosis each observation score at each Bielchowsky’s silver methods be used in shall be performed for the purpose of observation time. conjunction with more standard stains evaluating dose-response relationships. (C) The mean and standard deviation to determine the lowest dose level at All regions of the nervous system for each continuous endpoint at each which neuropathological alterations are exhibiting any evidence of observation time. observed. When new or existing data neuropathological changes should be (D) Results of statistical analyses for provide evidence of structural included in this analysis. Sections from each measure, where appropriate. alterations it is recommended that the all dose groups from each region will be (iii) All neuropathological GFAP immunoassay also be considered. coded and examined in randomized observations shall be recorded and A description of this technique can be order without knowledge of the code. arranged by test groups. This data may found in the reference under paragraph The frequency of each type and severity be presented in the following (g)(10) of this section. of each lesion will be recorded. After all recommended format: (A) Fixation and processing of tissue. samples from all dose groups including (A) Description of lesions for each The nervous system shall be fixed by in all regions have been rated, the code animal. For each animal, data must be situ perfusion with an appropriate will be broken and statistical analysis submitted showing its identification aldehyde fixative. Any gross performed to evaluate dose-response (animal number, sex, treatment, dose, abnormalities should be noted. Tissue relationships. For each type of dose- and duration), a list of structures samples taken should adequately related lesion observed, examples of examined as well as the locations, represent all major regions of the different degrees of severity should be nature, frequency, and severity of nervous system. The tissue samples described. Photomicrographs of typical lesions. Inclusion of photomicrographs should be postfixed and processed examples of treatment-related regions is strongly recommended for according to standardized published are recommended to augment these demonstrating typical examples of the histological protocols (protocols descriptions. These examples will also type and severity of the described in the references under serve to illustrate a rating scale, such as neuropathological alterations observed. paragraphs (g)(1), (g)(2), or (g)(11) of this 1+, 2+, and 3+ for the degree of severity Any diagnoses derived from 43860 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations neurological signs and lesions including of Toxicology and Environmental described in this section are intended to naturally occurring diseases or Health. 9:691–704 (1982). be used along with data from routine conditions, should be recorded. (6) Irwin, S. Comprehensive toxicity testing, to provide more (B) Counts and incidence of observational assessment: Ia. A accurate information on risk to the neuropathological alterations by test systematic quantitative procedure for immune system. The tests in this group. Data should be tabulated to assessing the behavioral physiological section do not represent a show: state of the mouse. comprehensive assessment of immune (1) The number of animals used in Psychopharmacologia. 13:222–257 function. each group and the number of animals (1968). (b) Source. The source material used in which any lesion was found. (7) Kinnard, E.J. and Watzman, N. in developing this TSCA test guideline (2) The number of animals affected by Techniques utilized in the evaluation of is the OPPTS harmonized test guideline each different type of lesion, the psychotropic drugs on animals activity. 870.7800 (June 1996 Public Draft). This locations, frequency, and average grade Journal of Pharmaceutical Sciences. source is available at the address in of each type of lesion. 55:995–1012 (1966). paragraph (j) of this section. (3) Evaluation of data. The findings (8) Meyer, O.A. et al. A method for the (c) Definitions. The following from the screening battery should be routine assessment of fore- and definitions apply to this section. evaluated in the context of preceding hindlimb grip strength of rats and mice. Antibodies or immunoglobulins (Ig) and/or concurrent toxicity studies and Neurobehavioral Toxicology. 1:233–236 are part of a large family of glycoprotein any correlated functional and (1979). molecules. They are produced by B cells histopathological findings. The (9) Moser V.C. et al. Comparison of in response to antigens, and bind evaluation shall include the relationship chlordimeform and carbaryl using a specifically to the eliciting antigen. The between the doses of the test substance functional observational battery. different classes of immunoglobulins and the presence or absence, incidence Fundamental and Applied Toxicology. involved in immunity are IgG, IgA, IgM, and severity, of any neurotoxic effects. 11:189–206 (1988). IgD, and IgE. Antibodies are found in The evaluation shall include (10) O’Callaghan, J.P. Quantification extracellular fluids, such as serum, appropriate statistical analyses, for of glial fibrillary acidic protein: saliva, milk, and lymph. Most antibody example, parametric tests for Comparison of slot-immunobinding responses are T cell-dependent, that is, continuous data and nonparametric assays with a novel sandwich ELISA. functional T and B lymphocytes, as well tests for the remainder. Choice of Neurotoxicology and Teratology. as antigen-presenting cells (usually analyses should consider tests 13:275–281 (1991). macrophages), are required for the (11) Pender, M.P. A simple method appropriate to the experimental design, production of antibodies. for high resolution light microscopy of including repeated measures. There may Cluster of differentiation (CD) refers to nervous tissue. Journal of Neuroscience be many acceptable ways to analyze molecules expressed on the cell surface. Methods. 15:213–218 (1985). data. These molecules are useful as distinct (12) Reiter, L.W. Use of activity (g) References. For additional CD molecules are found on different measures in behavioral toxicology. background information on this test populations of cells of the immune Environmental Health Perspectives. guideline, the following references system. Antibodies against these cell 26:9–20 (1978). should be consulted. These references (13) Reiter, L.W. and MacPhail, R.C. surface markers (e.g., CD4, CD8) are are available for inspection at the TSCA Motor activity: A survey of methods used to identify and quantitate different Nonconfidential Information Center, with potential use in toxicity testing. cell populations. Rm. NE–B607, Environmental Neurobehavorial Toxicology. 1— Immunotoxicity refers to the ability of Protection Agency, 401 M St., SW., Supplement. 1:53–66 (1979). a test substance to suppress immune Washington, DC, 12 noon to 4 p.m., (14) Robbins, T.W. Eds. Iversen, L.L., responses that could enhance the risk of Monday through Friday, except legal Iverson, D.S., and Snyder, S.H. A infectious or neoplastic disease, or to holidays. critique of the methods available for the induce inappropriate stimulation of the (1) Bennet, H.S. et al. Science and art measurement of spontaneous motor immune system, thus contributing to in the preparing tissues embedded in activity. Vol 7. Handbook of allergic or autoimmune disease. This plastic for light microscopy, with Psychopharmacology (Plenum, NY, section only addresses potential special reference to glycol methacrylate, 1977) pp. 37–82. immune suppression. glass knives and simple stains. Stain Natural Killer (NK) cells are large Technology. 51:71–97 (1976). § 799.9780 TSCA immunotoxicity. granular lymphocytes which (2) Di Sant Agnese, P.A. and De Mesy (a) Scope. This section is intended to nonspecifically lyse cells bearing tumor Jensen, K. Dibasic staining of large meet the testing requirements under or viral antigens. NK cells are up- epoxy sections and application to section 4 of TSCA. This section is regulated soon after infection by certain surgical pathology. American Journal of intended to provide information on microorganisms, and are thought to Clinical Pathology. 81:25–29 (1984). suppression of the immune system represent the first line of defense against (3) Edwards, P.M. and Parker, V.H. A which might occur as a result of viruses and tumors. simple, sensitive and objective method repeated exposure to a test chemical. T and B cells are lymphocytes which for early assessment of acrylamide While some information on potential are activated in response to specific neuropathy in rats. Toxicology and immunotoxic effects may be obtained antigens (foreign substances, usually Applied Pharmacology. 40:589–591 from hematology, lymphoid organ proteins). B cells produce antigen- (1977). weights and histopathology (usually specific antibodies (see the definition (4) Finger, F.W. Ed. Myers, R.D. done as part of routine toxicity testing), for ‘‘antibodies or immunoglobulins’’), Measuring Behavioral Activity. Vol. 2. there are data which demonstrate that and subpopulations of T cells are Methods in Psychobiology (Academic, these endpoints alone are not sufficient frequently needed to provide help for NY, 1972) pp.1–19. to predict immunotoxicity (Luster et al., the antibody response. Other types of T (5) Gad, S. A neuromuscular screen 1992, 1993 see paragraphs (j)(8) and cell participate in the direct destruction for use in industrial toxicology. Journal (j)(9) of this section). Therefore, the tests of cells expressing specific foreign Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43861

(tumor or infectious agent) antigens on major lymphocyte populations and T clear observation of each animal. The the cell surface. Cell subpopulations by flow cytometry, biological properties of the test (d) Principles of the test methods. (1) or a splenic NK cell activity assay to substance or toxic effects (e.g., In order to obtain data on the functional assess the effects of the test compound morbidity, excitability) may indicate a responsiveness of major components of on non-specific immunity shall be need for individual caging. the immune system to a T cell determined on a case-by-case basis, (B) The temperature of the dependent antigen, sheep red blood depending upon the outcome of the experimental animal rooms shall be at cells (SRBC), rats and/or mice1 shall be anti-SRBC assay. 22 ± 3°C. exposed to the test and control (e) Limit test. If a test at one dose level (C) The relative humidity of the substances for at least 28 days.2 The of at least 1,000 mg/kg body weight (or experimental animal rooms shall be animals shall be immunized by 2 mg/L for inhalation route of exposure) between 30 and 70%. intravenous or intraperitoneal injection using the procedures described for this (D) Where lighting is artificial, the of SRBCs approximately 4 days study produces no observable toxic sequence shall be 12 hrs light, 12 hrs (depending on the strain of animal) effects or if toxic effects would not be dark. prior to the end of the exposure. At the expected based upon data of structurally (E) Control and test animals shall be end of the exposure period, either the related compounds, then a full study maintained on the same type of bedding plaque forming cell (PFC) assay or an using three dose levels might not be and receive feed from the same lot. The enzyme linked immunosorbent assay necessary. Expected human exposure feed shall be analyzed to assure (ELISA) shall be performed to determine may indicate the need for a higher dose adequacy of nutritional requirements of the effects of the test substance on the level. the species tested and for impurities splenic anti-SRBC (IgM) response or (f) Test procedures—(1) Animal that might influence the outcome of the serum anti-SRBC IgM levels, selection—(i) Species and strain. These test. Rodents shall be fed and watered respectively. tests are intended for use in rats and/or ad libitum with food replaced at least (2) In the event the test substance mice. Commonly used laboratory strains weekly. produces significant suppression of the 4 shall be employed. All test animals (F) The study shall not be initiated anti-SRBC response, expression of shall be free of pathogens, internal and until the animals have been allowed an phenotypic markers for major external parasites. Females shall be adequate period of acclimatization or lymphocyte populations (total T and nulliparous and nonpregnant. The quarantine to environmental conditions. total B), and T cell subpopulations (T species, strain, and source of the The period of acclimatization shall be at helpers (CD4) and T cytotoxic/ animals shall be identified. least 1 week in duration. suppressors (CD8)), as assessed by flow (ii) Age/weight. (A) Young, healthy (2) Control and test substances. (i) cytometry, may be performed to animals shall be employed. At the The test substance shall be dissolved or determine the effects of the test commencement of the study, the weight suspended in a suitable vehicle. Ideally, substance on either splenic or variation of the animals used shall not if a vehicle or diluent is needed, it shall peripheral-blood lymphocyte ± exceed 20% of the mean weight for not elicit toxic effects or substantially populations and T cell subpopulations. each sex. alter the chemical or toxicological When this study is performed, the (B) Dosing shall begin when the test properties of the test substance. It is appropriate monoclonal antibodies for animals are between 6 and 8 weeks old. recommended that an aqueous solution the species being tested should be used. (iii) Sex. Either sex may be used in the should be used. If solubility is a If the test substance has no significant study; if one sex is known or believed problem a solution in oil may be used. effect on the anti-SRBC assay, a to be more sensitive to the test Other vehicles may be considered, but functional test for NK cells may be compound, then that sex shall be used. only as a last resort. performed to test for a chemical’s effect (iv) Numbers. (A) At least eight (ii) One lot of the test substance shall on non-specific immunity.3 For tests animals shall be included in each dose be used, if possible, throughout the performed using cells or sera from blood and control group. The number of duration of the study, and the research (ELISA or flow cytometry), it is not animals tested shall yield sufficient sample shall be stored under conditions necessary to destroy the animals, since statistical power to detect a 20% change that maintain its purity and stability. immunization with SRBCs at 28 days is based upon the interanimal variation Prior to the initiation of the study, there not expected to markedly affect the which may be encountered in these shall be a characterization of the test results of other assays included in assays. substance, including the purity of the subchronic or longer-term studies (these (B) To avoid bias, the use of adequate test compound and if technically tests are discussed in the reference randomization procedures for the feasible, the name and quantities of any under paragraph (j)(7) of this section). proper allocation of animals to test and known contaminants and impurities. The necessity to perform either a control groups is required. (iii) If the test or positive control quantitative analysis of the effects of a (C) Each animal shall be assigned a substance is to be incorporated into feed chemical on the numbers of cells in unique identification number. Dead animals, their preserved organs and or another vehicle, the period during 1 If absorption/distribution/metabolism/excretion tissues, and microscopic slides shall be which the test substance is stable in (ADME) data are similar between species, then identified by reference to the animal’s such a mixture shall be determined either rats or mice may be used for the test unique number. prior to the initiation of the study. Its compound in question. If such data are lacking, (v) Husbandry. (A) Animals may be homogeneity and concentration shall both species should be used. group-caged by sex, but the number of also be determined prior to the 2 Because there is a fairly rapid turnover of many of the cells in the immune system, 28 days is animals per cage shall not interfere with initiation of the study and periodically considered sufficient for the purposes of the anti- during the study. Statistically SRBC tests. 4 The study director shall be aware of strain randomized samples of the mixture 3 When these optional tests are included, the differences in response to SRBC. For example, if the shall be analyzed to ensure that proper phenotypic or NK cell analyses may be performed B6C3F1 hybrid mouse is used in the PFC assay, a at 28 days of exposure, or at a later timepoint if response of 800–1,000 PFC/106 spleen cells in mixing, formulation, and storage ADME data suggest that a longer exposure is more control mice should be the minimally acceptable procedures are being followed, and that appropriate. PFC response. the appropriate concentration of the test 43862 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations or control substance is contained in the approximately the same time each day, the vehicle on toxicity of, and mixture. and adjusted at intervals (weekly for penetration of the skin by, the test (3) Control groups. (i) A concurrent, mice, twice per week for rats) to substance should be taken into account. vehicle-treated control group is maintain a constant dose level in terms (3) The volume of application should required. of the animal’s body weight. be kept constant, e.g. less than 300 (ii) A separate untreated control group (iii) If the test substance is

(ii) The test system shall contain data (2) Number of animals showing signs of B lymphocytes. Vol. 1. Methods in on: of toxicity. Immunotoxicology (Wiley-Liss, Inc., (A) Species, strain, and rationale for (3) Number of animals dying. New York, 1995) pp. 71–108. selection of animal species, if other than (B) Individual animal data shall be (5) Ladics, G.S. and Loveless, S.E. Cell that recommended. presented, as well as summary (group surface marker analysis of splenic (B) Age, body weight data, and sex. mean data). lymphocyte populations of the CD rat (C) Date of death during the study or (C) Test environment including cage for use in immunotoxicological studies. whether animals survived to conditions, ambient temperature, Toxicology Methods. 4: 77–91 (1994). humidity, and light/dark periods. termination. (D) When inhalation is the route of (D) Date of observation of each (6) Ladics, G.S., Smith, C., Heaps, K., exposure, a description of the exposure abnormal sign and its subsequent and Loveless, S.E. Evaluation of the equipment and data shall be included as course. humoral immune response of CD rats follows: (E) Absolute and relative spleen and following a 2-week exposure to the (1) Description of test conditions; the thymus weight data. pesticide carbaryl by the oral, dermal, or following exposure conditions shall be (F) Feed and water consumption data, inhalation routes. Journal of Toxicology reported: when collected. Environmental Health. 42:143–156 (i) Description of exposure apparatus (G) Results of immunotoxicity tests. (1994). including design, type, volume, source (H) Necropsy findings of animals that (7) Ladics., G.S., Smith, C., Heaps, K., of air, system for generating aerosols, were found moribund and euthanized or Elliot, G.S., Slone, T.W., and Loveless, method of conditioning air, treatment of died during the study. S.E. Possible incorporation of an exhaust air and the method of housing (I) Statistical treatment of results, immunotoxicological functional assay the animals in a test chamber. where appropriate. for assessing humoral immunity for (ii) The equipment for measuring (i) Quality control. A system shall be hazard identification purposes in rats on temperature, humidity, and particulate developed and maintained to assure and standard toxicology study. Toxicology. aerosol concentrations and size should document adequate performance of 96:225–238 (1995). laboratory staff and equipment. The be described. (8) Luster, M.I., Portier, C., Pait, D.G., (2) Exposure data shall be tabulated study shall be conducted in compliance White, K.L., Jr., Gennings, C., Munson, and presented with mean values and a with the 40 CFR Part 792—Good A.E., and Rosenthal, G.J. Risk measure of variability (e.g., standard Laboratory Practice. assessment in immunotoxicology I. deviation) and include: (j) References. For additional Sensitivity and predictability of (i) Airflow rates through the background information on this test immune tests. Fundamental Applied inhalation equipment. guideline, the following references Toxicology. 18:200–210 (1992). (ii) Temperature and humidity of air. should be consulted. These references (iii) Actual (analytical or gravimetric) are available for inspection at the TSCA (9) Luster, M.I., Portier, C., Pait, D.G., concentration in the breathing zone. Nonconfidential Information Center, Rosenthal, G.J. Germolec. D.R., Corsini, (iv) Nominal concentration (total Rm. NE–B607, Environmental E., Blaylock, B.L., Pollock, P., Kouchi, amount of test substance fed into the Protection Agency, 401 M St., SW., Y., Craig, W., White, D.L., Munson, A.E., inhalation equipment divided by Washington, DC, 12 noon to 4 p.m., and Comment, C.E. Risk Assessment in volume of air). Monday through Friday, except legal Immunotoxicology II. Relationships (v) Particle size distribution, holidays. Between Immune and Host Resistance calculated mass median aerodynamic (1) Cornacoff, J.B., Graham, C.S., and Tests. Fundamental Applied diameter (MMAD) and geometric LaBrie, T.K. Eds. Burleson, G.R., Dean, Toxicology. 21:71–82 (1993). standard deviation (GSD). J.H., and Munson, A.E. Phenotypic (10) Temple, L., Kawabata, T. T., (vi) Explanation as to why the desired identification of peripheral blood Munson, A. E., and White, Jr., K. L. chamber concentration and/or particle mononuclear leukocytes by flow Comparison of ELISA and plaque- size could not be achieved (if cytometry as an adjunct to forming cell assays for measuring the applicable) and the efforts taken to immunotoxicity evaluation. Vol. 1. humoral immune response to SRBC in comply with this aspect of the section. Methods in Immunotoxicology (Wiley- rats and mice treated with (E) Identification of animal diet. Liss, Inc., New York, 1995) pp. 211–226. benzo[a]pyrene or cyclophosphamide. (iii) The test procedure shall include (2) Cunningham, A.J. A method of Fundamental Applied Toxicology. the following data: increased sensitivity for detecting single 21:412–419 (1993). (A) Method of randomization used. antibody-forming cells. Nature. (B) Full description of experimental 207:1106–1107 (1965). (11) Temple, L., Butterworth, L., design and procedure. (3) Djeu, Julie Y. Eds. Burleson, G.R., Kawabata, T.T., Munson, A.E., and (C) Dose regimen including levels, Dean, J.H., and Munson, A.E. Natural White, Jr., K.L. Eds. Burleson, G.R., methods, and volume. Killer Activity. Methods in Dean, J.H., and Munson, A.E. ELISA to (iv) Test results should include the Immunotoxicology. pp. 437–449 (1995). Measure SRBC Specific Serum IgM: following data: (4) Holsapple, M.P. Eds. Burleson, Method and Data Evaluation. Vol. 1. (A) Group animal toxic response data G.R., Dean, J.H., and Munson, A.E. The Methods in Immunotoxicology (Wiley- shall be tabulated by species, strain, sex, plaque-forming cell (PFC) response in Liss, Inc., New York, 1995) pp. 137–157. and exposure level for: Immunotoxicology: An approach to [FR Doc. 97–21413 Filed 8–14–97; 8:45 am] (1) Number of animals exposed. monitoring the primary effector function BILLING CODE 6560±50±F