Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity.

Mutation Research, 258 (1991) 259-283

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© 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1110/91/$03.50

M U T R E V 07307

Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity Kerry L. Dearfield 1, Angela E. Auletta 2, Michael C. Cimino 2 and Martha M. Moore 3 1 Health Effects Dicision, Office of Pesticide Programs, and 2 Health and Environmental Reciew DiL'ision, Office of Toxic Substances, U.S. Environmental Protection Agency, Washington, DC 20460 and ~ Genetic Toxicology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.A.) (Received 12 February 1991) (Accepted 30 May 1991)

Keywords: U.S. Environmental Protection Agency; Mutagenicity test procedures

Contents Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Office of Pesticide Programs (OPP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Revised initial battery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further actions beyond testing in initial battery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Office of Toxic Substances (OTS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Revised testing battery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further actions beyond testing in initial tier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing (protocol) guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A: Summary USEPA mutagenicity risk assessment guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix B: Scientific Advisory Panel and public comment process for OPP revised guideline . . . . . . . . . . . . . . . . . . . . . .

259 261 262 262 263 266 267 267 270 272 272 273 274

Summary OPP

This paper provides the rationale and support for the decisions the OPP will make in requiring and reviewing mutagenicity information. The regulatory requirement for mutagenicity testing to support a pesticide registration is found in the 40 CFR Part 158. The guidance as to the specific mutagenicity testing to be performed is found in the OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (referred to as the Subdivision F guideline).

Correspondence: Dr. K.L. Dearfield, USEPA, O P P / H E D (H7509C), 401 M St., S.W., Washington, DC 20460 (U.S.A.).

This manuscript has been reviewed by the Office of Pesticides and Toxic Substances and the Health Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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A revised Subdivision F guideline has been presented that becomes the current guidance for submitters of mutagenicity data to the OPP. The decision to revise the guideline was the result of close examination of the version published in 1982 and the desire to update the guidance based on developments since then and current state-of-the-science. After undergoing Agency and public scrutiny, the revised guideline is to be published in 1991. The revised guideline consists of an initial battery of tests (the Salmonella assay, an in vitro mammalian gene mutation assay and an in vivo cytogenetics assay which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation) that should provide an adequate initial assessment of the potential mutagenicity of a chemical. Follow-up testing to clarify results from the initial testing may be necessary. After this information as well as all other relevant information is obtained, a weight-of-evidence decision will be made about the possible mutagenicity concern a chemical may present. Testing to pursue qualitative a n d / o r quantitative evidence for assessing heritable risk in relation to human beings will then be considered if a mutagenicity concern exists. This testing may range from tests for evidence of gonadal exposure to dominant lethal testing to quantitative tests such as the specific locus and heritable translocation assays. The mutagenicity assessment will be performed in accordance with the Agency's Mutagenicity Risk Assessment Guidelines. The mutagenicity data would also be used in the weight-of-evidence consideration for the potential carcinogenicity of a chemical in accordance with the Agency's Carcinogen Risk Assessment Guidelines. In instances where there are triggers for carcinogenicity testing, mutagenicity data may be used as one of the triggers after a consideration of available information. It is felt that the revised Subdivision F guideline will provide appropriate, and more specific, guidance concerning the O P P approach to mutagenicity testing for the registration of a pesticide. It also provides a clearer understanding of how the OPP will proceed with its evaluation and decision making concerning the potential heritable effects of a test chemical. OTS As a result of recent information in the field of mutagenicity (the Williamsburg meeting, its precedents and sequelae), a modification to the Section 4 test scheme has been proposed. In the proposal, test schemes for gene mutation and chromosomal aberrations are combined. The revision comprises three tests in the initial tier: the Salmonella assay, an in vitro assay for gene mutation and an in vivo assay for chromosomal effects which may be either a bone marrow assay for chromosomal aberrations or the micronucleus assay. Since submission of this article for publication, the proposed OTS test scheme has been approved by OTS management. The old two scheme, four test scheme is no longer in effect, and will no longer be used in future T S C A Section 4 Test Rules. Under Section 5 of TSCA ("new" chemical, or Premanufacture Notice (PMN)), mutagenicity data are used for three purposes: (1) as part of exposure based testing; (2) to assess the potential of the P M N chemical to induce heritable genetic effects; and (3) as part of the weight-of-evidence that a chemical may be a potential carcinogen. As part of the exposure based testing program, the U S E P A has required testing of certain high volume chemicals with a two test battery of the Salmonella assay and a mouse micronucleus assay. In supporting a concern for potential carcinogenicity of a P M N chemical, the U S E P A generally cites data on an analogue which is known to be carcinogenic (i.e., demonstrated tumor forming ability in one or more animal studies). In such instances, mutagenicity data on the P M N chemical or on the analogues are used to lend support to the case for potential carcinogenicity. Where there is no analogue of the PMN chemical which has been tested for carcinogenicity, mutagenicity data alone are generally not considered sufficient to support a concern for potential carcinogenicity. Regulatory action under Section 5 is seldom, if ever, taken on the basis of mutagenicity data alone, especially on the basis of in vitro mutagenicity data.

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Introduction

Periodically, the U.S. Environmental Protection Agency (USEPA or Agency) reviews its mutagenicity testing guidelines to assess their effectiveness for evaluating genotoxicity data based on the current state of the science. The Office of Pesticide Programs (OPP) and the Office of Toxic Substances (OTS), which are both under the auspices of the USEPA's Office of Pesticides and Toxic Substances (OPTS), are now recommending changes in their current guidelines for mutagenicity testing. The science of mutagenicity testing has undergone considerable rethinking in the years since the current mutagenicity guidelines were proposed and initiated. In part, this rethinking has been the result of efforts by the USEPA's Gene-Tox Program to evaluate short-term tests for mutagenicity and related endpoints (Waters and Auletta, 1981) and the National Toxicology Program's studies of the ability of short-term in vitro tests to predict carcinogenicity (Tennant et al., 1987). Also, meetings such as the one sponsored by the USEPA in Williamsburg, VA in January, 1987 (Workshop on the Relationship Between Short-Term Test Information and Carcinogenicity) have spurred reconsideration of the role of short-term mutagenicity tests in toxicity testing (Auletta and Ashby, 1988; Kier, 1988). Many articles have been published over the past several years reflecting a myriad of opinions and viewpoints on the role(s) of mutagenicity testing (e.g., Ashby, 1986a,b; Garner and Kirkland, 1986; Gatehouse and Tweats, 1986; Lave and Omenn, 1986; Arni et al., 1988; Brockman and De Marini, 1988; Ennever and Rosenkranz, 1988; Legator and Harper, 1988). Many of these have dealt with short-term tests as predictors of carcinogenicity although some have emphasized heritable mutations as an endpoint of concern. In 1986, the Agency published its Mutagenicity Risk Assessment Guidelines (USEPA, 1986a). These guidelines deal with heritable mutation as a regulatory endpoint. They present a weight-ofevidence scheme for determining if an agent may be a potential human germ cell mutagen. Emphasis is placed upon a chemical's intrinsic mutagenic potential, its ability to reach the gonad and interact with germ cell DNA, and its ability to

induce heritable mutations in a mammalian species. The categories of evidence for chemical interaction in the gonad and of evidence that contributes to the weight-of-evidence evaluation for potential human germ cell mutagenicity are summarized in Appendix A. It is clear from the Mutagenicity Risk Assessment Guidelines that the USEPA intends to regulate chemicals based on risk of an adverse heritable effect in human germ cells. This is consistent with the evolution of Agency policy as detailed for example in USEPA (1978) and Hill (1979). It is noted that the USEPA has historically used mutagenicity information as part of its weight-of-evidence approach to discern the potential for human carcinogenicity. This is still a major use of mutagenicity data as detailed in the Agency's Carcinogen Risk Assessment Guidelines (USEPA, 1986b). However, it is emphasized that this is not the s01e use for mutagenicity data and that the Agency will evaluate mutagenicity data in terms of heritable risk. It is the policy of the Agency to periodically reevaluate its guidelines in accord with the current state of the science. By revising its mutagenicity guidelines at this time, the USEPA will be in concert with other countries and international bodies which have recently revised or are in the process of revising their mutagenicity guidelines (e.g., Canada (Health and Welfare Canada, 1986), the United Kingdom (Diggle and Fielder, 1989), and the EEC). The U.S. Food and Drug Administration is also revising its mutagenicity guidelines found in the "Red Book" (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food; V. Dunkel and D. Benz, personal communication). By revising both the OPP and the OTS guidelines at the same time, the USEPA intends to harmonize mutagenicity testing requirements of the two offices to the extent possible within the statutory limitations of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Toxic Substances Control Act (TSCA). The OPP and the OTS are also in the process of harmonizing their existing testing guidelines in the areas of chemical fate and health and ecological effects. It is anticipated that the end result of

262 this effort will be a set of OPTS testing guidelines that will be used by both offices. Differences which are necessary to meet the statutory requirements of each law will be detailed within each guideline. Once this effort is complete, the OPTS and the Organisation for Economic Cooperation and Development (OECD) guidelines will be compared to identify and reconcile differences to the degree possible. The thrust of this effort is to reach harmonization of testing methods and thereby ensure mutual acceptance of data both within the USEPA and between the U S E P A and the international community. This document will present the requirements for mutagenicity testing for submission of data to the U.S. Environmental Protection Agency's Office of Pesticide Programs and the Office of Toxic Substances. The rationale and support for the revised guidelines will also be presented and questions about the revision process and the guidelines themselves will be addressed.

Office of Pesticide Programs (OPP)

Background Section 3 (Registration of Pesticides) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) permits the Administrator of the USEPA to publish guidelines specifying the kinds of information required to support the registration of a pesticide with the OPP. It also states that revisions should be made from time to time (see Section 3 (c)(2) of FIFRA). Part 158 of the Code of Federal Regulations (40 CFR - Protection of Environment) is the regulation that determines what toxicity testing must be performed and when to perform such tests in support of a pesticide registration under FIFRA. The OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (subsequently referred to as Subdivision F guideline; present version from USEPA, 1982) provides guidance on how to implement the Part 158 requirements. This paper will discuss the revisions to the OPP's Subdivision F mutagenicity guideline and the rationale for those revisions. The OPP is revising Part 158 to allow for the generation of data more appropriate for making a sound regulatory decision. The revisions to Sub-

division F would therefore also be timely in anticipating upcoming revisions to Part 158. Part 158 - Data Requirements for Registration - specifies the types and minimum amounts of data required in order to make regulatory judgments about the risks of pesticide products. The current requirements concerning mutagenicity testing are detailed in Part 158. It states that mutagenicity testing is required for all general use patterns of pesticides (including terrestrial, aquatic, greenhouse, forestry, domestic outdoor and indoor for both food crop and nonfood uses). The technical grade of the active ingredient is the test substance to be used for testing. One test in each of three categories (gene mutation, structural chromosomal aberrations, and other genotoxic effects) is minimally required. It does not appear that the upcoming revisions to Part 158 will change the requirement that mutagenicity testing be performed for all general use patterns of pesticides. However, the general requirement for testing in the three categories mentioned above will be eliminated in favor of testing in specified assays as detailed in the Subdivision F guideline revision (see below). The OPP mutagenicity guideline is found in Series 84 of the Subdivision F guidelines. It is divided into two sections: (1) 84-1: Purpose and General Recommendations for Mutagenicity Testing and (2) 84-2: Mutagenicity Tests. Section 84-1 is fairly self-explanatory in describing the purpose and general recommendations for mutagenicity testing, such as which substance to test, standards for metabolic activation, and control materials. Section 84-2 states that a minimum of three mutagenicity tests be performed, one each in the categories of gene mutations, structural chromosomal aberrations and other genotoxic effects (the last including numerical chromosome anomalies and direct DNA damage and repair). Lists of representative tests that would satisfy each category are provided from which particular tests may be chosen. Although no specific protocol guidelines are provided, references to some standards are mentioned, such as those of the Gene-Tox Program from the USEPA's OTS. Current practice by the OPP, however, is to utilize the protocol guidelines issued by the OTS that are found in the 40 CFR Part 798 - Health

263 Effects Testing Guidelines (for discussion, see section on Testing (protocol) guidelines).

Rer'ised initial battery A major criticism of the 1982 published Subdivision F guideline is that it provides little guidance regarding the choice of tests to be performed for a particular chemical or class of chemicals. The OPP agrees with this assessment and believes it would be appropriate to provide more specific guidance as to what tests should be performed. To that end, it has been suggested that chemicals to be submitted to OPP for registration purposes should be tested in a defined battery of mutagenicity tests (Dearfield, 1989). The results of such testing would provide the U S E P A with information to be used in assessing the potential mutagenic hazard of chemical agents subject to regulation under FIFRA. The Subdivision F guideline revision has undergone rigorous Agency and public examination (for details and discussion of various issues relating to this guideline, see Appendix B). The decisions and rationale that culminated in the current revised Subdivision F guideline are presented here and in Appendix B. The revised Subdivision F guideline is projected to be officially published in 1991. Submitters of mutagenicity data to the OPP are advised to take these new guidelines

into account when planning future submissions to the OPP. The tests that will be included in the OPP initial battery are as follows (also seen in Fig. 1): (1) Salmonella typhimurium reverse mutation assay. (2) Mammalian cells in culture forward gene mutation assay allowing detection of point mutations, large deletions and chromosomal rearrangements, such as: (a) mouse lymphoma L5178Y cells, thymidine kinase (tk) gene locus, maximizing assay conditions for small colony expression and detection; or, (b) Chinese hamster ovary (CHO) or Chinese hamster lung fibroblast (V79) cells, hypoxanthineguanine phosphoribosyl transferase (hgprt) gene locus, accompanied by an appropriate in vitro test for clastogenicity; or, (c) Chinese hamster ovary (CHO) cells strain AS52, xanthine-guanine phosphoribosyl transferase (xprt) gene locus. (3) An in vivo assay for chromosomal effects using either: (a) metaphase analysis (aberrations); or, (b) a micronucleus assay. For an initial assessment of mutagenic activity, the in vivo assays are most often performed in rodent bone marrow. Use of other species or

C U R R E N T OPP MUTAGENICITY TEST GUIDELINE

Salmonella

+

In Vitro + G e n e Mutation *

In Vivo Bone Marrow C y t o g e n e t i c s • Aberrations or • Micronucleus

* In Vitro Gene Mutation (choice): (a) Mouse lymphoma L5178Y cells, tk locus, small and large colonies (b) Chinese hamster ovary cells strain AS52 (c) Chinese hamster ovary (CHO) or Chinese hamster lung fibroblasts (V79) cells + appropriate in vitro test for clastogenicity Fig. 1. Current OPP mutagenicity test guideline.

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other target organs should be discussed with the OPP prior to testing. According to the Agency's Mutagenicity Risk Assessment Guidelines (USEPA, 1986a), there are several mutagenesis endpoints of concern including point mutations (i.e., submicroscopic changes in the base sequence of DNA) and structural or numerical chromosome aberrations. Structural aberrations include deficiencies, duplications, insertions, inversions, and translocations of parts of chromosomes, whereas numerical aberrations are gains or losses of whole chromosomes (e.g., trisomy, monosomy) or sets of chromosomes (haploidy, polyploidy). The revised OPP battery will identify those agents which induce point (or gene) mutations a n d / o r structural chromosome aberrations. Since there is no assay that is currently validated and routinely available for testing agents that may induce numerical chromosome aberrations (e.g., aneuploidy events; Dellarco et al., 1986), such an assay is not included in the proposed battery. Until such time as a reliable test is developed and validated, the Agency will deal with agents which may induce numerical chromosomal aberrations on a case-by-case basis. Despite this obvious limitation, the OPP believes that the revised battery will provide sufficient information about chemical activity to allow a reasonable assessment of potential mutagenic hazard. The first test in the revised OPP testing battery is the Salmonella/mammalian microsomal assay. The Salmonella assay is a reverse mutation assay which employs several specific tester strains for the detection of point mutations. Since the genetics of each tester strain have been well-defined, it is possible to identify the specific type of genetic effect (i.e., base substitution, frameshift mutation) induced by agents which are active in this system (Ames et al., 1973; Maron and Ames, 1983). In addition to genetic characterization, the Salmonella assay has several other advantages which make it a logical choice for inclusion in a testing battery. This assay is easy to perform and is used routinely throughout the world and in most laboratories which perform genetic toxicology testing (Farrow et al., 1984). As a result of this wide-spread use, it is both well-validated and has an extensive data base of tested chemicals

(Kier et al., 1986; Auletta and Kier, in preparation). It is extremely useful for detecting intrinsic mutagenicity of many classes of biologically active chemicals, especially ones that appear to act via an electrophilic mechanism (e.g., Rinkus and Legator, 1979; Ashby and Tennant, 1988). Although there are disagreements about the assay's precise ability to accurately predict chemical carcinogenicity, it nonetheless provides useful information in predicting carcinogenic, as well as mutagenic, potential of chemical agents. Both Kier (1988) and Auletta and Ashby (1988) recommend inclusion of the Salmonella assay in any screening program to set priorities for further testing by identification of potentially mutagenic and carcinogenic chemical agents. The Salmonella assay is included in the OPP battery for all of these reasons. Although the Salmonella assay is a primary test for gene mutations, it was felt that it should not be the sole test for this endpoint. The Agency, according to its Guidelines for Mutagenicity Risk Assessment (USEPA, 1986a), places greater weight on tests conducted in eukaryotes than in prokaryotes and in mammalian species rather than in submammalian species in conducting a hazard evaluation of a chemical. Major differences between mammalian cells and bacterial cells, such as membrane structures, DNA repair capabilities and the organization and complexity of mammalian genomes, suggested that it was necessary to have a mammalian system included in the battery (Fox, 1988). Furthermore, there are chemicals which give negative results in the Salmonella assay which are mutagenic when tested in a mammalian celt culture assay for gene mutations (Arlett and Cole, 1988). Finally, the results of the NTP study on the use of short-term tests as predictors of carcinogenicity (Tennant et al., 1988; Auletta and Ashby, 1988; see also Benigni, 1989, for cluster analysis of NTP data) show that the L5178Y mouse lymphoma cell assay and the sister chromatid exchange assay appear to be sensitive to a different subset of chemicals than the Salmonella assay and the in vitro cytogenetics assay. The OPP concluded, therefore, that the combination of the Salmonella assay and a mammalian cell culture assay for gene mutation would provide more information than

265 that obtained from the Salmonella assay alone. This additional information may provide a better idea of the mechanism of mutagenic activity, a refinement of possible mutagenicity concern, a n d / o r a basis for further testing. In selecting a mammalian cell culture assay for inclusion in the OPP battery, primary consideration was given to the L5178Y mouse lymphoma assay. This has generated a great deal of comment during the Subdivision F Agency and public examination period (see Appendix B). In selecting an in vitro mammalian gene mutation assay, an important consideration was the ability of the chosen assay to provide maximum information on the genotoxicity of the test chemical. Recent advances in the understanding of the types of genetic events detectable by mammalian cell assays indicate that two assays, the L 5 1 7 8 Y / T K +/mouse lymphoma assay and the C H O AS52 assay, detect chemicals capable of inducing both point mutations and chromosomal events (Hsie, 1987; Stankowski and Tindall, 1987; Moore et al., 1989; Applegate et al., 1990). Use of an assay capable of detecting a broad range of genetic events provides the most information and is thus preferred. The mouse lymphoma assay is the better characterized of the two assays with regard to the types of genetic damage detected. By using a combination of molecular analysis and banded karyotype analysis, research has shown that tk mutants appear to include presumed point mutations (no visible alteration in karyotype or Southern blot pattern), total tk gene deletions, mitotic nondisjunction, translocation, homologous mitotic recombination, and gene conversion (Applegate et al., 1990). In humans, genetic lesions including rearrangements, additions and deletions of genetic material result in heritable disorders and are associated with neoplasia. The AS52 assay, a relatively new modification of the standard Chinese hamster ovary ( C H O ) / h g p r t assay, appears capable of detecting clastogenic events not detected by the standard C H O assay (Hsie, 1987; Stankowski and Tindall, 1987). Because of the ability to detect other genotoxic effects in addition to point mutations, both of these assays are appropriate for use in the initial assessment of the mutagenicity of a test compound.

An in vivo cytogenetics assay was chosen as the assay of choice for determining effects on chromosomes. This may either be metaphase analysis for chromosomal aberrations (excluding SCE formation) or a micronucleus assay, both conducted in rodent bone marrow. These tests have been performed routinely for many years and each has a substantial data base of tested chemicals (Preston et al., 1981; Heddle et al., 1983; Mavournin et al., 1990). Other organ or tissue sites may be considered (e.g., liver, lymphocytes, spleen), particularly if knowledge about the test chemical provides support for the selection of other organs/tissues (e.g., in George et al., 1989, the rat liver carcinogen 2-nitropr0Pane was negative in the bone marrow micronucleus assay, but positive in a liver micronucleus assay; similarly, Schmezer et al., 1990, show that N-nitrosodibenzylamine induces micronuclei in rat liver, but not in rat bone marrow). Since the Agency's Guidelines for Mutagenicity Risk Assessment place greater weight on results from in vivo tests than in vitro tests (USEPA, 1986a), it was felt that the chromosomal aberration assay should be performed in vivo, thus allowing for such factors as intact in vivo metabolism, repair capabilities, pharmacokinetic factors (e.g., biological half-life, absorption, distribution, excretion), and target specificities (see, e.g., Legator and Harper, 1988). This initial battery was designed for use with chemicals of unknown genotoxic potential. This battery should provide much useful information about mutagenic potential without imposing unduly burdensome testing requirements upon the registrant. It is designed to satisfy the minimum regulatory requirement for initial mutagenicity testing. However, alternative tests may be proposed if such testing is based on knowledge of the test chemical. The OPP intends to be responsive to unique testing considerations for specific chemicals or classes of chemicals. If other tests or special testing conditions appear more appropriate for specific chemicals, then the submitter or the OPP may propose a discussion of these points before testing is initiated. These overall changes in the mutagenicity testing scheme were made in response to the criticism that the current guideline, with its lists of representative tests, was too broad in scope to

266 provide guidance for many submitters. By proposing this initial battery of tests, the OPP accomplishes two purposes: (i) it provides a defined set of mutagenicity tests to be performed on chemicals to be submitted for registration, and (ii) it ensures the generation of a body of data on which to base decisions about either the need for further testing a n d / o r the degree of concern about the potential mutagenicity of the test agent. In addition to the initial battery, other provisions are provided to enhance the information submitted to the OPP for registration of chemicals. If other tests for endpoints that may be predictive of mutagenicity are performed in addition to the initial battery, these results must also be submitted to the OPP. A reference list of all studies/papers known to the submitter concerning the mutagenicity of the test chemical is also to be submitted. This does not need to be a totally exhaustive effort, but one that reasonably captures most of the relevant studies on the test compound. Registrants most likely perform this function in their own deliberations (or should) and it should not be an additional burden. Submission of other relevant data is encouraged (e.g., metabolism, distribution studies, reproductive studies - - which in many cases are already required by the Part 158 Toxicology Data Requirements). This additional information may provide better insight into the significance and interpretation of mutagenicity test results, and would greatly facilitate the OPP's effort to provide a timely and more accurate assessment of the submitted chemical.

Further actions beyond testing in initial battery Confirmatory testing or other relevant information may be required to provide clarification of equivocal or discordant results among the tests initially submitted to the OPP. This would provide information that may further clarify the potential genotoxic hazard of the test chemical. For example, additional in vivo cytogenetics testing may be required to address such concerns as target tissue/organ or species specificity, differences in m e t a b o l i s m or distribution, or structure-activity relationship (SAR) considerations.

Results from the initial battery and confirmatory testing (if performed) are reviewed along with all other available relevant information before decisions on subsequent regulatory action are made. The purpose of the initial testing is to assess inherent mutagenicity for the purpose of hazard identification. If no mutagenicity hazard is identified from the available information, further action may not be necessary. However, if additional information becomes available which suggests a mutagenicity hazard, then the decision to take no further action may be reconsidered. Further testing to determine if a chemical may induce heritable mutation in mammals will be performed, if necessary, in accordance with the Agency's Guidelines for Mutagenicity Risk Assessment (USEPA, 1986a). When evaluating a chemical for the potential to induce heritable mutations, all available data including mutagenicity test data; exposure data; SAR considerations; studies of mechanism of action; pharmacokinetic and metabolism information; the results of testing for reproductive effects, target organ specificity, subchronic and chronic effects; and the ability of the chemical to reach the germ cell and interact with gonadal DNA will be considered. Once the available data have been reviewed, the Agency may decide that no further testing is warranted. However, if the weight-of-evidence suggests further testing, that testing may involve cytogenetic testing in spermatogonia a n d / o r spermatocytes of rodents, dominant lethal testing, or testing for other evidence of chemical interaction with mammalian germ cells. Results of such tests, if positive, would provide evidence that the chemical in question has the potential to induce heritable mutation in mammals (presumably including humans). The need for additional testing to support a quantitative risk assessment would depend upon both available mutagenicity data and other relevant considerations such as human exposure levels, chemical use patterns and release to the environment. When the qualitative evidence using this approach suggests a potential hazard for heritable mutagenic effects, appropriate tests for quantifying heritable risk shall be performed. Currently, these tests are the specific locus test (either visible or biochemical) and the heritable transloca-

267

tion test, both performed in rodents. Upon completion of appropriate tests for quantifying heritable risk, a quantitative risk assessment will be performed. It is recognized that quantitative mutagenicity risk assessment is still a fledgling area, but previous efforts to perform quantitative risk assessment will be examined (e.g., see UNSCEAR, 1977; Selby, 1979; BEIR, 1980; Ehling, 1988; Rhomberg et al., 1990) in order to identify and apply appropriate methods to quantify the risk due to the test chemical under examination. Mutagenicity test results will also be considered in decisions about the carcinogenicity of the test chemical. If a chemical has been tested for carcinogenicity, available mutagenicity data will be used along with the carcinogenicity test(s) results as part of the weight-of-evidence approach for classifying the chemical in accordance with the Agency's Guidelines for Carcinogen Risk Assessment (1986b). When carcinogenicity testing is conditionally required in accordance with the Part 158 Toxicology Data Requirements, evidence of chemical mutagenicity may provide the basis to require a carcinogenicity study for that chemical. Office of Toxic Substances (OTS)

Background The Toxic Substances Control Act (TSCA) provides the U S E P A with the authority "to regulate commerce and protect human health and the environment by requiring testing and necessary use restrictions on certain chemical substanc e s . . . " This authority supplements other existing laws, such as the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), the Clean Air Act, the Clean Water Act, and the Occupational Safety and Health Act. TSCA is designed to fill the gap in the government's authority to test and regulate chemicals. In TSCA, the term "chemical" encompasses a wide variety of organic and inorganic substances manufactured or imported for industrial uses, such as dyes, pigments, lubricant additives, chemical intermediates, synthetic fibers, structural polymers, coatings - - essentially any commercial chemical except those used as drugs, food additives, cosmetics, pesticides and certain other uses.

TSCA has two main regulatory features: (1) acquisition of sufficient information by EPA to identify and evaluate the potential hazards from chemical substances; and (2) regulation of the production, use, distribution and disposal of such substances when necessary. Section 4(a) of TSCA gives the USEPA authority to require the testing of chemicals if unreasonable risk to health or the environment is suspected. To require testing, EPA must find that: (1) the chemical may present an unreasonable risk or a significant potential for exposure; (2) there are insufficient data available with which to perform a reasoned risk assessment; and (3) testing is necessary to generate such data (and is not already underway). A testing requirement is promulgated in a Test Rule, which must: (1) identify the substance to be tested and the tests to be conducted; (2) provide (or reference) guidelines for performance of the tests; and (3) specify a reasonable period of time for completion of testing. In the past, the OTS required that chemicals which were subject to mutagenicity testing under Section 4(a) of TSCA be tested independently for both gene mutation and chromosomal aberrations. The test schemes were designed in 1979; each scheme was composed of three tiers. In the gene mutation scheme (Fig. 2), testing began with the Salmonella assay. A chemical which was negative in Salmonella was tested in an in vitro assay for gene mutation in mammalian cells in culture. A positive in either assay triggered a Drosophila sex-linked recessive lethal assay. A positive in the sex-linked recessive lethal assay triggered a visible specific locus assay. The chromosomal effects test scheme (Fig. 3) began with an in vitro cytogenetics assay. A negative in vitro assay triggered an in vivo bone marrow cytogenetics assay (either metaphase analysis for aberrations or micronuclei). A positive in either assay triggered a rodent dominant lethal assay. A positive rodent dominant lethal assay triggered a rodent heritable transiocation assay. The first four assays in these two test schemes (Salmonella, in vitro gene mutation, and in vitro and in vivo cytogenetics assays) were selected to optimize the detection of intrinsic mutagenic potential. If all were negative, no further testing was

268

FORMER OTS GENE MUTATION TEST SCHEME

Salmonella w/wo a~vation

In Vitro

NEGATIVE

NEGATIVE ~

Gene Mutation

No Further

Testing

w/wo activation POSmVE POSITIVE

Bioassay Trigger NEGATIVE

=- Drosophila SLR POSmME

Bioassay

,

Trigger

Specific Locus Fig. 2. Former OTS gene mutation test scheme.

required. The next two tests (Drosophila sex-linked recessive lethal and rodent dominant lethal assays) demonstrate the ability of the chemical to reach the gonad of the intact organism and to interact with germ cell DNA. If both assays were negative, no further testing was required. Agents which were positive in the sex-linked recessive

lethal assay were to be tested further in the mouse visible specific locus test; those which were positive in the rodent dominant lethal assay were to be tested in the rodent heritable translocation assay. Agents which were positive in either of these two assays (specific locus a n d / o r heritable translocation assays) would be presumed to be

FORMER OTS CHROMOSOME MUTATION TEST SCHEME

In Vitro ~E Cytogenetics w/wo activation Posmw Bioassay

v

v

In Vivo Cytogenetics

~.~VE

Posmw

Trigger Dominant Lethal

~7~TWE

Posrr~E

Bioassay Trigger

Heritable Translocation Fig. 3. F o r m e r O T S c h r o m o s o m e m u t a t i o n test scheme.

No Further Testing J

269

potential human mutagens as outlined in the U S E P A ' s mutagenicity risk assessment guidelines. Several factors entered into the choice of tests in this scheme. First, it was deemed necessary to test independently for both gene mutation and chromosomal effects in the event that agents existed which were specifically gene or chromosomal mutagens. Second, it was decided to use only those tests which measure defined genetic endpoints. Tests such as the sister chromatid exchange or unscheduled D N A synthesis assays, which either do not measure a defined genetic endpoint or for which the specific endpoint of concern is not known, were not included in the generic test scheme. Such tests have been included in specific Test Rules if existing data suggested that they might be sensitive indicators of genotoxicity for the chemical in question. The OTS also uses short-term mutagenicity test data as an indicator of potential carcinogenicity. In 1982, certain key tests in the Section 4(a) test schemes were selected as triggers to a 2-year carcinogenicity bioassay. At that time, a positive response in certain tests served as a trigger to a cancer bioassay: (1) A positive response in both the Salmonella and the Drosophila sex-linked recessive lethal assays; or (2) A single positive response in: (a) the m a m m a l i a n cell culture assay for gene mutation; (b) the in vitro assay for chromosomal aberrations; or (c) the in vivo assay for chromosomal effects (either chromosomal aberrations or micronucleus formation). To date, the OTS has not required a 2-year bioassay based solely on the results of short-term mutagenicity tests. Section 5 of T S C A requires that manufacturers and importers of " n e w " chemicals submit a Premanufacturing Notification (PMN) to the U S E P A 90 days before beginning the manufacture or import of the new substance. By " n e w " chemicals, T S C A means those chemicals not appearing on the Inventory of Existing Commercial Chemicals, which was originally compiled in 1977 and is continuously updated.

Section 5 does not require that submitters conduct toxicity testing prior to submission of the PMN, only that they submit such data which are either in their possession or readily obtainable by them. Therefore, the OTS has developed techniques for mutagenicity hazard assessment which can be used in the presence of few or no test data on the substance itself. This approach involves the following three components: (1) Evaluation of available toxicity data on the PMN chemical, if any. (2) Evaluation of test data available on substances which are analogues of the PMN chemical, or of data which are available on key potential metabolites or analogues of the metabolites. (3) Use of knowledge and judgment of scientific assessors in the interpretation and integration of the information developed in the course of the assessment. In general, mutagenicity data are used for three purposes under Section 5: (1) as part of exposure based testing; (2) to assess the potential of the PMN chemical to induce heritable genetic effects; and (3) as part of the weight-of-evidence that a chemical may be a potential carcinogen. Recently, as part of the exposure based testing program, where the U S E P A requires testing of certain high volume chemicals which reach a trigger for occupational or consumer use exposure, the requirement has been for a two test battery of the Salmonella assay and a mouse micronucleus assay. Because of the nature of the PMN assessment process, which relies heavily upon the use of analogue data, and because of limitations in the size of the data base of chemicals tested for heritable genetic effects, concern for a chemical's ability to induce heritable gene or chromosomal mutations is rarely supportable under Section (5) Therefore, the principal use of mutagenicity data under Section 5 has been as part of the weightof-evidence for carcinogenicity. In supporting a concern for potential carcinogenicity of a PMN chemical, the U S E P A will generally cite data on an analogue which is known to be carcinogenic (i.e., demonstrated tumor forming ability in one or more animal studies). In such instances, mutagenicity data on the PMN chemical or on the analogue(s) are used to lend

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support to the case for potential carcinogenicity. Where there is no analogue of the PMN chemical which has been tested for carcinogenicity, mutagenicity data alone are generally not considered sufficient to support a concern for potential carcinogenicity. Regulatory action is seldom, if ever, taken on the basis of mutagenicity data alone, especially on the basis of in vitro mutagenicity data. Mutagenicity data have been required as part of several Section 5(e) notices. Initially, these requirements were primarily for Salmonella test data. For certain classes of chemicals, the requirement was for in vitro gene mutation data; specifically for data from the L 5 1 7 8 Y / T K +/ mouse lymphoma system. More recently, the U S E P A has begun requiring certain manufacturers to submit data from a battery of tests which has included the Salmonella assay and an in vivo assay for chromosomal effects, especially the micronucleus assay. Where an appropriate carcinogenic analogue has been tested in the same assays as those required for the PMN chemical, this agent is generally included in the test as a positive control chemical. In some cases, where activity of the analogue chemical in short-term tests was not known, the U S E P A has required the simultaneous testing of both the PMN chemical and the analogue. Positive results for both the PMN chemical and the analogue are used to support the weight-of-evidence that the PMN chemical may be a carcinogen. In these instances, a 2-year bioassay of the PMN chemical or the use of protective equipment to limit exposure are generally required. Negative results for the PMN chemical, in the face of positive results for the analogue, are taken as an indication that the PMN chemical is probably non-carcinogenic, or in cases where the analogy may have been doubtful, that the analogue was not appropriate. In either case, concern for potential carcinogenicity is lessened as a result of mutagenicity data and a 2-year bioassay is generally not considered necessary. In those instances where the analogue chemical is inactive in short-term tests for mutagenicity, negative results for the PMN chemical do not alleviate concern for potential carcinogenicity.

Because of the cost of conducting a long-term cancer bioassay, such a requirement is often interpreted by the regulated industry as a de facto ban. Chemicals subjected to the requirement for a long-term cancer bioassay are often withdrawn by the submitter because they cannot support the cost of testing. The U S E P A hopes that judicious and reasonable use of short-term testing will increase in the Section 5 process and that this increase in the use of short-term testing will reduce the number of chemicals subject to a bioassay.

Revised testing battery As stated above, in 1982 the OTS designated certain key tests in the TSCA Section 4 mutagenicity test schemes as direct triggers to a cancer bioassay. Since then, data from the Agency's Gene-Tox Program and the NTP Testing Program have stimulated discussion on the predictive ability of some short-term tests. These data were the subject of discussion at an Agency sponsored Workshop on the Relationship Between ShortT e r m Test Information and Carcinogenicity ("the Williamsburg meeting") (Auletta and Ashby, 1988; Kier, 1988). As a result of data presented at this workshop and subsequently published in the scientific literature (Tennant et al., 1987), the OTS has proposed revising its mutagenicity test schemes and the tests which serve as triggers to a cancer bioassay. These revisions are shown in Fig. 4. The major change found in the revised test scheme is in the first tier where it is proposed to combine the test schemes for gene mutation and chromosomal aberrations. The revision includes three tests for the first tier: the Salmonella assay, an in vitro assay for gene mutation and an in vivo assay for chromosomal effects which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation. Under the provisions of TSCA Section 4 the U S E P A has the right to require a cancer bioassay immediately based upon " m a y present an unreasonable risk" criteria such as structure-activity relationship (SAR) information a n d / o r production or release data. If accepted after proposal and review of public comment, the revised scheme would be used in instances where the U S E P A

271 makes a policy decision to trigger carcinogenicity testing from mutagenicity test data. In these instances, the situation vis-a-vis the use of shortterm tests to trigger a bioassay would be as follows. A positive response in all three first tier tests the in vivo assay for chromosomal effects o r a positive response in the in vitro gene mutation assay and the in vivo assay for chromosomal effects would lead directly to a 2-year bioassay. Although there would still be an automatic trigger to a bioassay, it would be dependent upon a minimum of two positive responses, at least one of which must be in an in vivo assay. Any other combination of responses, including a single positive response in any one assay, o r a positive response in b o t h the Salmonella assay and the in vitro assay for gene mutation, would result in a "data review." The data review, which would occur before a decision was made to require further testing, would consider all available information including other test results, structure-activity relationships, production volume and exposure figures. o r a positive in the Salmonella assay a n d

The in vitro cytogenetics assay would no longer be part of the test scheme. At one time, the OTS considered removing the in vitro assays for gene mutation from the test scheme as well. However, that position has been reconsidered in the light of unofficial comment on that proposal and as a result of work which has been performed since the Williamsburg meeting. For now, these assays would remain part of the test scheme but they would no longer serve as single test triggers to a bioassay. The Drosophila sex-linked recessive lethal assay would be replaced by other assays to assess interaction with mammalian gonadal DNA. These other assays include some combination of unscheduled D N A synthesis, chromosomal aberrations, sister chromatid exchange and alkaline elution, all in testicular cells, and the dominant lethal assay. The Drosophila test would no longer serve as a trigger to a bioassay. The OTS proposed revisions to the first tier of the Section 4 mutagenicity test schemes are not substantially different from the OPP initial battery. The OTS has chosen not to designate a

CURRENT OTS MUTAGENICITY TEST SCHEME Salmonella

+

+ In Vitro Gene Mutation

Interaction with Gonadal DNA

Specific Locus • Visible or • Biochemical

In Vivo Bone Marrow Cytogenetics • Aberrations or • Micronucleus

Dominant Lethal

Heritable Translocation

Fig. 4. Current OTS mutagenicitytest scheme.

272 specific test as the assay of choice for in vitro gene mutation studies preferring to make a decision about use of a particular assay at the time of promulgation of a Section 4 test rule. However, the OTS agrees that for routine screening purposes, chemicals should be tested in either the LS178Y mouse lymphoma or the C H O AS52 assays. It is anticipated that no further testing would be required for the majority of chemicals which are negative in all three first tier tests. However, where exposure data, structure-activity relationships or other factors indicate it is warranted, these agents may also be subject to a data review and subsequent testing in a cancer bioassay.

Further actions beyond testing in initial tier Once intrinsic mutagenicity has been identified, the revised scheme requires assay(s) that assess the ability of the chemical to interact with D N A in the gonad. This agrees with the principles presented in the U S E P A Mutagenicity Risk Assessment Guidelines (see Appendix A); i.e., in vivo tests are weighted more heavily than in vitro tests, mammalian systems more heavily than nonmammalian systems, and germ cell assays more heavily than somatic cell assays. Examples of assays that assess interaction with gonadal D N A are in vivo unscheduled D N A synthesis (UDS), alkaline eiution (AET), sister chromatid exchange or chromosomal aberration assays in testicular tissues, or the rodent dominant lethal assay. There are presently no practical assays to evaluate potential gene mutagenicity in vivo in the mammalian gonad. Results in testicular UDS and A E T assays show good correlation with the specific locus assay, based upon review of the U S E P A Gene-Tox data base (Bentley et al., 1991). This scheme has already been used on one occasion in the Test Rule process. It has been presented to the scientific community on several occasions in open meetings, and has met with general approval. Removal of these assays from the test scheme would necessitate an immediate trigger to a specific locus assay from a positive response in an in vitro assay (Salmonella assay or mammalian cells in culture), a requirement deemed to be too costly in the absence of some evidence of gonadal effect.

In addition, although present evidence indicates that chemicals positive in the heritable translocation assay are positive in the specific locus assay as well, there is no assurance that all gonadal chromosome mutagens will be gonadal gene mutagens. There is evidence from testing in somatic cells that such agents may exist (e.g., acrylates; Moore et al., 1989). At present, there are no changes in the third tier of the scheme, except that the gene mutation scheme now includes a mouse biochemical specific locus test as an alternative to the mouse visible specific locus test. Agents which are positive in the second tier gonadal D N A assay(s) may be tested in either specific locus assay; the choice will be left to the regulated industry. It is anticipated that a data review, similar to that mentioned above for the cancer bioassay, will be performed for those agents which may require testing in either the specific locus assay or the rodent heritable translocation assay.

Testing (protocol) guidelines One of the most frequent inquiries the OPP receives addresses the lack of specific guidance on how the OPP expects the mutagenicity tests to be performed. It is not the purpose of the Subdivision F guideline revision effort to formulate specific protocols for each mutagenicity test. Such guidelines already exist, drafted by the OTS (USEPA, 1985, 1987). The OPP felt it is appropriate at this time to provide specific guidance to registrants for the conduct of mutagenicity tests. Thus, the OPP now recognizes the published OTS protocol guidelines as ones the OPP will follow in reviewing submitted studies. Adoption by the OPP of the OTS protocol guidelines allows harmonization between the two offices; a common set of protocol guidelines for mutagenicity testing is therefore provided in response to mutagenicity testing requirements. Guidance for the performance of mutagenicity testing is found in the 40 C F R Part 798 - Health Effects Testing Guidelines, Subpart F - Genetic Toxicity (issued annually in the CFR; also published in USEPA, 1985, 1987). These guidelines have already undergone extensive public review and are periodically revised when appropriate to

273

reflect the current state of the science for each test. Submitters therefore should be aware of updated protocols before initiating testing for submission to the OPP and the OTS. Where no specific guideline is given, submitters are advised to discuss with the Agency proposed methods for the chosen test to ensure suitability of the test and acceptability of data. All testing submitted to the Agency needs to be performed under the requirements of the G o o d Laboratory Practice (GLP) Standards. The G L P standards for T S C A requirements are found in the 40 C F R Part 792 - Good Laboratory Practice Standards (USEPA, 1989a). The G L P standards that satisfy F I F R A requirements are found in the 40 C F R Part 160 - G o o d Laboratory Practice Standards (USEPA, 1989b). The G L P standards are harmonized between both offices.

Acknowledgements We would like to express our sincere thanks to Drs. Irving M a u e r , R i c h a r d Hill, D a v i d Jacobson-Kram, Reto Engler, Penelope FennerCrisp, Vicki Dellarco, Lawrence Valcovic and Michael Waters for their many contributions to this effort and for critical reading of this material.

Appendix A: Summary USEPA mutagenicity risk assessment guidelines The U S E P A published its Mutagenicity Risk Assessment Guidelines in 1986. These guidelines provide guidance for assessing evidence for chemical interaction in the gonad and evidence that would contribute to the weight-of-evidence for potential human germ cell mutagenicity. They are briefly summarized here. For full discussion, refer to the published guidelines (USEPA, 1986a). According to the guidelines, there are two categories of evidence for chemical interaction in the gonad: (1) Sufficient evidence of chemical interaction is given by the demonstration that an agent interacts with germ cell D N A or other chromatin constituents, or that it induces such endpoints as unscheduled D N A synthesis (UDS), sister chromatid exchanges (SCE), or chromosomal aberrations in germinal cells.

(2) Suggestive evidence includes the finding of adverse gonadal effects such as sperm abnormalities following acute, subchronic or chronic toxicity testing, or findings of adverse reproductive effects such as decreased fertility, which are consistent with the chemical's interaction with germ cells. There are eight categories of evidence that contribute to the weight-of-evidence for potential human germ cell mutagenicity. These are, in order of decreasing strength-of-evidence: (1) Positive data derived from human germ cell mutagenicity studies. (2) Valid positive results from studies on heritable mutational events (of any kind) in mammalian germ cells. (3) Valid positive results from mammalian germ cell chromosome aberration studies that do not involve transmission from one generation to the next. (4) Sufficient evidence for a chemical's interaction with mammalian germ cells, together with valid positive mutagenicity test results from two assay systems, at least one of which is mammalian (in vitro or in vivo). The positive results may both be for gene mutation or both for chromosome aberrations; if one is for gene mutations and the other for chromosome aberrations, both must be from mammalian systems. (5) Suggestive evidence for a chemical's interaction with mammalian germ cells, together with valid positive mutagenicity evidence from two assay systems as described under 4, above. Alternatively, positive mutagenicity evidence of less strength than defined under 4, above, when combined with sufficient evidence for a chemical's interaction with mammalian germ cells. (6) Positive mutagenicity test results of less strength than defined under 4, combined with suggestive evidence for a chemical's interaction with mammalian germ cells. (7) Although definitive proof of non-mutagenicity is not possible, a chemical could be operationally classified as a non-mutagen for human germ cells if it gives valid negative test results for all endpoints of concern. (8) Inadequate evidence bearing on either mutagenicity or chemical interaction with mammalian germ cells.

274 Appendix B: Scientific Advisory Panel and public comment process for OPP revised guideline Before a revision to an OPP guideline is finalized and issued as the guidance for testing under F I F R A , it must be presented to the OPP's Scientific Advisory Panel (SAP) and the public for comments. The SAP is an external peer review group that meets periodically to provide expert analysis and opinions on scientific decisions the OPP will make. Once the SAP and the public provided comments on the proposed guidelines, the comments were analyzed and addressed before issuing the final Subdivision F guideline. A proposed mutagenicity guideline was presented before the SAP on September 29, 1989 and the public comment period was opened with the issuance of a Federal Register notice that announced both the SAP meeting time and the availability of appropriate documents for examination (USEPA, 1989c). The Federal Register notice also provided for a public comment period from August 25, 1989 to September 12, 1989; this was subsequently extended to October 31, 1989. The proposed revision to the Subdivision F guideline was rigorously discussed during the SAP meeting and in comments received from the public. These considerations weighed heavily in the formulation of the final Subdivision F guideline revision. The single issue that generated the most discussion was the original proposal to rely exclusively on the mouse lymphoma assay as the in vitro mammalian gene mutation assay. As noted in the final revised guideline and in response to the SAP and the public comments, this preference had been changed to allow a choice to satisfy this testing requirement. Other aspects of the Subdivision F guideline were also discussed and are detailed below. The report of the SAP's recommendations addressed nine general issues on the F I F R A proposed revised mutagenicity testing guidelines (report issued via the SAP Executive Secretary, October 16, 1989; to obtain copies, address inquiries to the SAP Executive Secretary, Office of Pesticide Programs). The nine issues were: (1) L5178Y as the preferred assay for gene mutations in mammalian cells. (2) Omission of the in vitro cytogenetics assay.

(3) Dosing limits. (4) Basis for positive controls. (5) Three tests versus two tests. (6) Negative (nonsolvent) controls in in vitro tests. (7) Aroclor as the preferred enzyme inducer. (8) Mouse peripheral blood micronucleus (MN) test. (9) Test results to support proposed test scheme. A total of 14 public commentors submitted written comments on the proposed mutagenicity testing guidelines. In addition to the nine general issues raised above, the public comments raised six additional issues for consideration. These were: (1) Requirement for confirmation of in vitro assays. (2) Discontinue "other genotoxic effects" category from current scheme. (3) Provide more guidance on test protocols. (4) Guidance to assist in carcinogenicity classification and mathematical modelling. (5) Use of biochemical specific locus assay. (6) Timing of the review of genetic toxicity tests. Specific responses and comments to the SAP and public comments are presented below. L5178Y as the preferred assay for gene mutations in mammalian cells The preference for the mouse lymphoma assay to satisfy testing with the mammalian gene mutation assay was the issue that generated the greatest amount of comment from the public as well as from the SAP. Among the points brought forward, as discussed by the SAP, were: (a) " . . . the assay responds to certain chemicals that are not recognized at this time to be mutagenic in other systems. For this reason, responses to chemicals or conditions of unknown or unverified mutagenicity in L5178Y cannot be concluded, with a sufficient degree of certainty, to be evidence of mutagenicity or of potential hazard." (b) " . . . the SAP foresees difficulties in establishing the assay in new laboratories and obtaining consistent results." (c) "Studies of chemicals that induce an increase in T F T resistant clones may yield im-

275 portant information on the mechanism by which such clones arise, but until a better understanding of this range of mechanisms is achieved, the L5178Y assay is not recommended for E P A ' s preferred test for mutation in cultured mammalian cell." (d) " I t is r e c o m m e n d e d that E P A not include this assay in its testing guidelines; however, in the future, if an adequate scientific rationale can be developed for its inclusion, it should appear with alternative mammalian assays listed before it." The OPP feels that all of these issues can be adequately addressed in supporting a preference for the mouse lymphoma assay in testing for mammalian gene mutations. It is noted that in the public comments, there was some support for retaining the mouse lymphoma assay preference. The support for this preference is detailed here. It is well established that the mouse lymphoma assay responds to known mutagenic agents and that trifluorothymidine (TFT) resistant clones identified by this assay may arise as the result of genetic changes induced by interaction between such agents and the genetic material (Clive et at., 1983; Mitchell et al., in preparation). It has also been demonstrated that some chemicals that apparently should not be mutagenic have produced activity in this assay (e.g., see Cifone et al., 1987; Wangenheim and Bolcsfoldi, 1988). In many instances, this activity in this assay could be attributed to extreme test conditions, e.g., effects of pH, osmotic imbalances, and concentrations of $9 mix used. However, this is not unique to the mouse lymphoma assay. Other in vitro assays experience similar problems when non-physiological conditions are employed (high osmotic pressure, pH, etc.); for example, increased chromosomal aberrations due to increased osmotic pressure (Galloway et al., 1987). There are two additional considerations. (1) Test results must be carefully evaluated with common sense with regard to what is occurring in the assay under the test conditions. This consideration should apply for all assays, but historically seems to be minimized when evaluating data from the mouse lymphoma assay. In some instances,

the evaluation of test data considered a result "positive" when in actuality it may have not have been a biologically significant increase. This is a matter of interpretation that is being directly addressed in upcoming publications from leading experts in the mouse lymphoma assay and should provide guidance for consistent evaluations in the future (Gene-Tox Phase IIl evaluation of the mouse lymphoma assay, Mitchell et al., in preparation; Clive et al., in preparation). This guidance will be available to the OPP when mouse lymp h o m a results need to be evaluated. (2) The decision on interpreting a clear positive response in the mouse lymphoma assay when there are negative responses in other assays will be dealt with on a case by case basis. In these instances, all available data will be considered before a decision is reached on the potential genotoxicity of the test chemical. There is no reason to dismiss as irrelevant a clear positive response in the mouse lymphoma assay when there are negative responses in other assays. The SAP commented on the difficulty in establishing the assay and obtaining consistent results. The mouse lymphoma assay is one of the major mammalian gene mutation assays in routine use (Farrow et al., 1986) and is performed in laboratories all over the world. As with any assay being newly established in a laboratory, it is not unusual to experience problems with start-up and routine performance. Also, assay systems are continually evolving and questions about proper methodology for many of the "standard" assays may arise over time. Similarly, techniques for the performance of the mouse lymphoma assay are evolving and being refined for a better characterization of activity by test chemicals. However, the need for consistency in the performance of a " r o u t i n e " assay is understandable and efforts have been made for optimizing the performance of this assay (Clive et al., in preparation). The Health Effects Testing Guidelines will include a new, individual guideline for the mouse lymp h o m a assay which will be published in the near future (Cimino and Auletta, 1990). The types of genetic damage detected in the mouse lymphoma assay have been well characterized; the U S E P A itself has funded a major research effort towards this end. As detailed above,

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tk mutants a p p e a r to include presumed point mutations (no visible alteration in karyotype or Southern blot pattern), total tk gene deletions, mitotic nondisjunction, translocation, homologous mitotic recombination, and gene conversion (Applegate et al., 1990). As new techniques in this area become available, it is expected that the range of genetic effects detected by the mouse lymphoma assay will be further defined. Therefore, in comparison to other mammalian gene mutation assays, the mouse lymphoma assay may detect a wider range of genetic effects, thus making it a more powerful tool for assessing genetic activity by a test chemical. The genetic locus used in the mouse lymphoma assay may account for its ability to detect a broad range of effects. In contrast, in the standard C H O / h g p r t assay, the hgprt locus is found in a hemizygous state (on the X chromosome). The nature of this locus may limit the recovery of multi-locus deletions and also prohibit the induction of homologous mitotic recombination. Agents which exert such effects would therefore not be detected. The tk locus is heterozygous (located on an autosome) and thus should be able to tolerate multi-locus deletions. It also has been demonstrated that mitotic recombination a n d / o r gene conversion can be detected in L5178Y cells (Applegate et al., 1990). Moore et al. (1989) have reviewed the literature and made direct comparisons of the two assays. This analysis clearly demonstrates the inefficiency of the C H O / h g p r t assay for detecting a number of chemicals that are detected by the mouse lymphoma assay (e.g., chemicals acting via a clastogenic mechanism). The C H O AS52 assay, a relatively new modification of the standard C H O / h g p r t assay, appears capable of detecting some of the genetic events not detected by the standard C H O assay (Hsie, 1987; Stankowski and Tindall, 1987). The OPP recognizes this assay as a possible alternative to the mouse lymphoma L5178Y assay. Another gene mutation assay, the T K - 6 / t k assay, also appears capable of detecting a range of events similar to that detected in the mouse lymphoma assay (Little et al., 1987). However, it has not been used for chemical screening and thus is not r e c o m m e n d e d as a part of a routine test battery at this time. The SAP and public comments suggested that

if the mouse lymphoma assay is retained in the initial battery, it should not be the preferred mammalian gene mutation assay. The rationale behind the selection of the mouse lymphoma assay as the preferred assay was its ability to detect a wider range of genetic effects than the other "routine" mammalian gene mutation assays, such as the C H O or V79 assays using the hgprt gene locus. For this reason the OPP stresses detection of small colonies. This will be reflected in the proposed Health Effects Testing Guidelines which will specify conditions for optimum detection of small colonies. It appears that small and large colonies induced by test chemicals represent different genetic effects (Moore et al., 1985a,b) and these should be quantified to take full advantage of the assay. The standard C H O / h g p r t assay has been shown to not detect many clastogenic test chemicals (Moore et al., 1989) and would not provide as complete information on the genetic activity of many test chemicals. There was a consistent recommendation m a d e in the public c o m m e n t s that the C H O / h g p r t assay be acceptable if it is accompanied by an in vitro assay for chromosomal aberrations. In the revised Subdivision F guideline, the OPP accepts the SAP recommendation that the mouse lymphoma assay not be the exclusive assay for the mammalian gene mutation assay. The OPP is providing a choice for this data requirement, but with the understanding that there will not be a loss of the amount of information that can be obtained from these in vitro assays.

Omission of the in vitro cytogenetics assay Concern was raised by the SAP and some public comments about the omission of the in vitro cytogenetics assay from an initial testing battery. Specifically, the SAP "considers the in vitro cytogeneties test plus the Salmonella test to be an adequate pair of tests to detect in vitro mutagenic activity." Furthermore, "if a scientific rationale for the use of an in vitro mammalian cell gene mutation assay in addition to the Salmonella assay can be established, it is recommended that the in vitro cytogenetic assay be indicated as an equal replacement."

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In response, the O P P felt that the Salmonella test plus the mammalian gene mutation assays as described above are adequate to detect in vitro mutagenic activity. The O P P believes it is important to supplement the prokaryotic Salmonella test with an eukaryotic gene mutation test. It is recognized that the in vitro cytogenetics assay can provide valuable information concerning the mutagenic potential of a test chemical. However, there are several considerations that led the OPP to prefer the mammalian gene mutation assays (mouse lymphoma or C H O AS52) as the in vitro m a m m a l i a n test. The genetic damage induced in an in vitro cytogenetics assay may not be compatible with cell survival. The gene mutation assays count colonies that consist of viable cells. Mutations compatible with cell survival may ultimately be more relevant to risks that can be passed on to future generations. Additionally, the in vitro cytogenetics assay may not be as quantitative as the gene mutation assay; for example, up to 200 cells are scored for cytogenetics v e r s u s 10 6 for gene mutations. Furthermore, there is a test for structural chromosomal aberrations in the initial battery, the in vivo cytogenetics assay. This was selected for the reasons provided above (in OPP section). With the in vivo test in the initial battery, it was felt that the in vitro cytogenetics test would not be as necessary as the other tests in the initial battery. Also, historically, whenever there has been a positive result in the in vitro cytogenetics test, an in vivo cytogenetics assay was performed to assess the relevance of the in vitro result to the in vivo situation. Therefore, it was decided to perform an in vivo cytogenetics assay in the initial battery. This preference has been voiced elsewhere (Legator and Harper, 1988; Shelby, 1988). Concerns have been expressed about o r g a n / t i s sue specificity in vivo and the possibility that only limited information from the in vivo cytogenetics assay would be obtained. However, decisions to perform additional in vivo testing with other target organs or tissues can be made using all available information on the test chemical.

Dosing limits The SAP states " I n both in vitro and in vivo assays, excessive dose levels may lead to re-

sponses that are not the result of a direct interaction between the test chemical and the genetic material of the test organism. For this reason, it may be advisable for the guidelines to recommend upper limits of test concentrations." Examples such as toxicity parameters and molarity considerations are provided by the SAP. In the absence of any other limiting signs, arbitrary limits of 5000 ~ g / m l in vitro and 5000 m g / k g in vivo are suggested as common upper limits. It is agreed that excessive dose levels and concentrations may lead to responses which are not due to direct genetic effects by the test chemical. The suggested limits are believed to be reasonable. Dose limits due to other factors such as toxicity are discussed for each genetic test in the Health Effects Testing Guidelines (see Testing (protocol) guidelines above). For this reason, it is not necessary to reiterate them in the Subdivision F guideline (which is not a protocol guideline).

Basis for positive controls The SAP states "the section of the guidelines dealing with positive controls needs to be written more clearly in order to explain the purpose of such controls and to distinguish between recommendations for in vitro and in vivo tests. The rationale for recommending that positive controls use the same solvent as the test agent and the same route of exposure should be clearly stated." It is agreed that this section (positive controls in the General Recommendations section) of the Subdivision F guideline could be made clearer and a distinction made between in vitro and in vivo testing circumstances. However, it should be r e m e m b e r e d that these are general recommendations and specific guidance for positive controls for each genetic test is found in the Health Effects Testing Guidelines (see Testing (protocol) guidelines above). Positive control compounds should be selected to demonstrate the sensitivity of the test system and, for in vitro assays, the functioning of the metabolic activation system. In several instances, the revised Health Effects Testing Guidelines for mutagenicity will recommend that a positive control be selected to ensure the detection of a minimal response. This would test not only the functioning of the test system, but also the ability

278

of the investigator to perform the assay and analyze the resulting data. For in vitro assays, the positive control compound is usually administered in a solvent consistent with the properties of the compound. Ideally, but not necessarily, the solvent for the positive control chemical will be the same as that used for the test chemical. The positive control may be selected in accordance with the performing facility's historical data base to allow comparison with previous performance of the assay. For in vivo assays, where it is feasible, the positive control should be administered in the same vehicle and by the same route as the test chemical. However, it is recognized that there are circumstances where this would not be feasible and positive controls administered with a different vehicle and by a different route would be acceptable. Three tests L'ersus two tests

There were several public comments requesting information on the use of three tests in the OPP initial test battery instead of the use of only two tests (Salmonella and in vivo cytogenetics being the two most often mentioned). The SAP preferred the three test option: " T h e Panel discussed the adequacy of recommending only two tests (Salmonella plus in vivo chromosomal aberrations) as being sufficient for pest control products. While there is some evidence that a priori these tests may be sufficient to identify most mutagenic chemicals, they do not provide sufficient assurance, given the possibility of human exposure, that such chemicals are not mutagenic. Accordingly, a minimum of two in vitro and an in vivo test provide a more appropriate level of assurance of a lack of potential hazard." This recommendation is reflected in the Subdivision F guideline revision. NegatiL,e (nonsoluent) controls in in vitro tests

The draft Subdivision F guideline in its General Recommendations section recommends that assays include both a solvent control and, where applicable, a nonsolvent negative control. The SAP states that "although useful information is sometimes obtained from negative controls, fully adequate tests need not include such a control. It

is, therefore, recommended that negative (nonsolvent) controls not be included in the guidelines." It is agreed that a negative (nonsolvent) control is not necessary for a fully adequate test.

Aroclor as the preferred e n z y m e inducer

The draft Subdivision F guideline in its General Recommendations section states that "a metabolic activation system should be incorporated into any test system that does not provide adequate metabolic capabilities." It suggests that rat liver extracts have the greatest usage and provides an example of such an activation system, e.g., post-mitochondrial fractions prepared from Aroclor 1254 induced rat livers. Generally, it is necessary to induce various enzyme activities in order to maximize the possibility of converting a compound requiring metabolism into potential genotoxic products, especially in genetic tests that are of short duration where an underestimation or a lack of possible metabolic activation by endogenous processes may occur. The SAP states, with regard to the inducing agent, "it is known that the species of origin and concentration of liver homogenate, as well as the chemical used as an enzyme inducer, can influence the mutagenic response of in vitro tests. It is r e c o m m e n d e d that the E P A indicate that these variables should be optimized where possible so that inappropriate in vitro metabolic activation mixtures are not used." It is agreed that variables in the production of exogenous metabolic activation systems should be optimized for the chemical under test; e.g., enzymes necessary for appropriate activation/detoxification pathways should be induced. While there are viable alternatives to its use (e.g., phenobarbital and /3-naphthoflavone), Aroclor appears to be the most widely used inducing agent. Among the reasons for its wide-spread use are: convenience, e.g., only one injection necessary for rats to be induced; commercial availability; relatively wide spectrum of induced enzymes; and a large historical data base with which to compare results. Aroclor was therefore mentioned as a primary example of an inducing agent. However, the OPP does not intend to suggest Aroclor is the only appropriate inducing agent. The use of different inducing agents with appro-

279

priate justification and validation would be allowed.

Mouse peripheral blood micronucleus (MN) test It was suggested in the public comments (and has been suggested to the OPP in the past) that results from mouse peripheral blood micronucleus tests performed as part of a subchronic study (e.g., 30-90 days exposure) be used to satisfy the requirement for an in vivo cytogenetics assay. The SAP states "the majority of available data support this proposal, but in view of the limited data base, the SAP considers it premature to include this proposal in the guidelines." While the Agency agrees that the technology to perform the mouse peripheral blood micronucleus tests exists, it also recognizes that the data base is limited. Whereas the current protocol guideline in the Health Effects Testing Guidelines (see Testing (protocol) guidelines above) does not cover the peripheral blood assay, a proposed revision to the micronucleus test guideline will provide guidance on the performance of this test. However, in keeping with the current state of the science for this assay as reflected in the SAP reservation, the protocol guideline revision for the micronucleus test will emphasize the bone marrow as the prime target tissue. The development of a larger data base for the peripheral blood micronucleus assay is encouraged. Test results to support proposed test scheme The SAP suggests that an analysis of the available data from short-term genetic toxicity tests, germ cell mutagenicity tests, and rodent carcinogenicity tests should be performed to support the revised Subdivision F guideline. For the OPP to perform such a detailed analysis on results from short-term genetic toxicity tests, germ cell mutagenicity tests and rodent carcinogenicity tests would be time and resource prohibitive. Numerous analyses of this nature have already been performed (e.g., the U S E P A Gene-Tox Program and the NTP efforts). The OPP has considered these analyses in formulating its current guideline. Requirement for confirmation of in ritro assays Good scientific practice suggests that every

experiment should be verified in an independent repeat assay. The Subdivision F guideline, however, does not deal with this question directly. The issue of repeat assays for both in vitro and in vivo tests is addressed in the protocol guidelines for each particular genetic test (see Testing (protocol) guidelines).

Discontinue "other genotoxic effects" category from current scheme It is recognized that the category "other genotoxic effects" Covers a broad category of genetic endpoints and that no specific guidance is given for choice of tests in this category. This category, as well as the other two categories, gene mutations and chromosomal aberrations, are being discontinued in favor of more specific guidance for test selection. For full discussion, see Revised initial battery (OPP section). Pror,ide more guidance on test protocols The Subdivision F guideline is not a protocol guideline; it provides guidance as to the types of tests necessary to support a pesticide registration, when tests should be performed relative to each other, and how the OPP will use mutagenicity data towards a decision for heritable risk. It is recognized that the OPP in the past has not provided specific protocol guidance on how to perform mutagenicity tests. With this guideline revision, the OPP formally adopts the Health Effects Testing Guidelines, Subpart F, Genetic Toxicity (in Code of Federal Regulation 40, Part 798; also in USEPA, 1985, 1987) as the protocol guidelines (see Testing (protocol) guidelines above for full discussion). Guidance to assist in carcinogenicity classification and mathematical modelling The use of mutagenicity data in the weight-ofevidence approach for classifying the carcinogenicity of a test compound is outside the purview of the Subdivision F guideline. While this guideline states that mutagenicity data will be used in a classification decision, specific guidance on the use of mutagenicity data in the classification and modelling of carcinogenicity test data is found in the U.S. Environmental Protection Agency's Guidelines for Carcinogen Risk Assessment

280 (USEPA, 1986b). The OPP follows these guidelines. The Carcinogen Risk Assessment guidelines themselves are periodically examined for revisions; comments such as this would be appropriate at those times.

Use of the biochemical specific locus assay One of the public comments expressed a reservation about the use of data from the mouse biochemical specific locus assay (MBSL) for quantitative risk assessment. The specific locus test (Russell et al., 1984) is one of the tests that could be required if information suggests that a chemical may present a heritable genetic risk and if the Agency believes quantitation of that risk is necessary for regulatory action. Historically the Agency has recommended the mouse visible specific locus assay (MVSL) (Russell et al,, 1981) for this purpose. However, the MVSL is not widely available. Also, because of the relatively small number of chemicals targeted for testing in the MVSL, laboratories have not set aside the resources to develop the assay and to establish a historical control data base. As a viable alternative to the use of the MVSL, the U S E P A examined the use of the MBSL to detect chemicals which elicit gene mutations in mammals (USEPA, 1988). With both tests, mutations can be detected at two important stages of development in the male sperm cell, the spermatogonial stem cell stage and the postspermatogonial stage. The U S E P A has concluded that the MBSL is as acceptable as the MVSL for determining the potential of a chemical to elicit heritable gene mutations in mammals (for full rationale, see USEPA, 1988, 1990). A testing guideline for the MBSL has been published and incorporated into the Health Effects Testing Guidelines (USEPA, 1990). Although the testing guidelines for both specific locus assays do not specify the use of more than one dose level, it should be noted that for the purposes of quantitation for risk assessment, more than one dose level will be required.

Timing of the review of genetic toxicity tests The proposed guideline describes a decision point after review of available genetic toxicity tests. At that time, the OPP will determine if there is a concern for genetic toxicity a n d / o r if

additional testing is necessary. Some commentors suggest " . . . that EPA review the initial battery of genetic toxicity studies as soon as the reports are available. This would allow OPP to review the data while long-term feeding studies are being performed and if additional genetic toxicology studies are required, then they could be performed concurrently with other studies." They recommend that the OPP "review the genetic toxicology results as soon as they are available rather than waiting for an entire registration package to be completed." It is outside the purview of the Subdivision F guideline revision effort to set times and deadlines for the submission and review of genetic toxicology tests. The times for submission of genetic toxicology tests, as well as for all toxicity testing, are determined by F I F R A , Registration Standards for each chemical, or specific regulatory actions taken by the OPP, as in the registration of new chemicals, Special Reviews, and Data Call-In Notices. For further information on the timing of submissions, one should contact the appropriate OPP Division (e.g., Registration Division, Special Review and Reregistration Division). Once the genetic toxicology tests have been submitted, the OPP will review the results and make decisions according to its mandates. Decisions from this review process should be timely and not cause undue delay in the regulatory process.

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