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The L5178Y/tk+/- (-)3.7.2C mouse lymphoma assay (MLA) which detects mutations affecting the heterozygous thymidine kinase (tk) locus is capable of responding to chemicals acting as clastogens as well as point mutagens. Improvements in the assay to enhance detection of this spectrum of genetic events are summarized, and criteria for evaluating the data are defined. Using these criteria, the Phase III Work Group reviewed and evaluated literature containing MLA results published from 1976 through 1993. The data base included 602 chemicals of which 343 were evaluated as positive, 44 negative, 18 equivocal, 54 apparently inappropriate for evaluation in this test system with the published protocols, and 142 that were inadequately tested, and thus a definitive call could not be made. The overall performance of the assay is summarized by chemical class, and the outcome of testing 260 chemicals in the MLA is compared with Gene-Tox and National Toxicology Program evaluations of rodent carcinogenesis bioassay results for the same chemicals. Based on the Work Group's evaluation of published MLA data for chemicals that were considered adequately tested, it is concluded that for most chemicals the L5178Y/tk+/- mouse lymphoma assay is eminently well suited for genotoxicity testing and for predicting the potential for carcinogenicity.

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The former U.S. EPA OPPT tiered test scheme for heritable gene mutations included the Drosophila sex-linked recessive lethal (SLRL) test in which positive results triggered the mouse specific locus (MSL) test. However, review of available literature indicated that the evaluation of mutations in the germ cells of this insect is not a good predictor of the risk of heritable gene mutations in mammals. The database contained 29 compounds for which there were conclusive MSL test results in either spermatogonial and/or postspermatogonial cells. Results in the SLRL test were available for 27 of those compounds. Of the 24 SLRL-positive chemicals, only 13 (54%) induced heritable mutations in mice; the three SLRL-negative compounds were nonmutagenic in mouse germ cells. The overall concordance between the two tests was 59%. In contrast, results of unscheduled DNA synthesis (UDS: 18 chemicals) and alkaline elution (AE: 14 chemicals) assays in rodent testicular cells following in vivo exposure correlated well with results in the MSL test (83% and 86%, respectively). MSL test results in spermatogonia and postspermatogonia were also compared separately to the SLRL, UDS, and AE assays. The concordances for the two cell types in the SLRL relative to the MSL test were 36% and 79%, respectively, indicating that the SLRL test is extremely poor in predicting heritable gene mutations in mammalian spermatogonia. Concordances for UDS and AE assays relative to MSL test results in spermatogonia (53% and 54%, respectively) and postspermatogonia (91% and 100%, respectively) were greater. Based on these analyses, the U.S. EPA OPPT has revised its tiered test scheme using assays for interaction with gonadal DNA (e.g., UDS and AE) in place of the SLRL test.

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New requirements for chemicals subject to mutagenicity testing from the U.S. Environmental Protection Agency (USEPA) are discussed. Also detailed are two categories in the 1986 Mutagenicity Risk Assessment Guidelines.

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The U.S. Environmental Protection Agency's Gene-Tox Program is a multiphased effort to review and evaluate the existing literature in assay systems available in the field of genetic toxicology. The first phase of the Gene-Tox Program selected assay systems for evaluation, generated expert panel reviews of the data from the scientific literature, and recommended testing protocols for the systems. Phase II established and evaluated the database of chemical genetic toxicity data for its relevance to identifying human health hazards. The ongoing phase III continues reviewing and updating chemical data in selected assay systems. Currently, data exist on over 4000 chemicals in 27 assay systems; two additional assay systems will be included in phase III. The review data are published in the scientific literature and are also publicly available through the National Library of Medicine TOXNET system. The review and analysis components of Gene-Tox comprise 45 published papers, and several others are in preparation. Differences that have been observed between Gene-Tox and National Toxicology Program databases relative to the sensitivity, specificity, accuracy, and predictivity of genetic toxicity data compared to carcinogenesis data are ascribable to differences between the two databases in chemical selection criteria, testing protocols, and chemical class distributions.

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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). 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 and/or 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 OPP 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.(ABSTRACT TRUNCATED AT 400 WORDS)

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The present status of the applicability of mammalian cell gene mutation assays in the safety evaluation of industrial chemicals is evaluated from the industrial and regulatory point of view, with emphasis being placed on the CHO/HGPRT and mouse lymphoma tk +/- assays. The CHO/HGPRT assay was concluded to be a highly specific assay, but it might be less sensitive to mutagens that mainly induced large deletions. The mouse lymphoma assay was concluded to be sensitive, but it might have a lower specificity due to experimental artifacts such as pH and osmolality changes. Mammalian gene mutation assays, when conducted within their limitations, are concluded to be valuable in safety evaluation, providing results complementary to the Ames test and cytogenetic assays.

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Results of the 1986 Genetic Toxicology Association's survey of industrial, government, contract, and academic laboratories on the status of several assays in genetic toxicology are presented below. 1. The most commonly used assay was the Salmonella typhimurium/mammalian microsomal (Ames) assay, which was used by 83% of all respondents. 2. The next five (5) most commonly used assays were in vitro cytogenetics (72%), in vivo cytogenetics (59%), CHO HGPRT gene mutation (55%), the micronucleus assay (53%), and L517BY gene mutation (45%). 3. The assay showing the greatest percentage increase in routine use was the micronucleus assay which went from 14% in 1984 to 34% in 1986, an increase of 20%. 4. Other assays which increased in routine use were CHO HGPRT mutation (+18%); in vitro cytogenetics (+14%); L5178Y gene mutation (+9%), and the Ames assay (+5%). 5. Routine use of in vitro UDS assays declined by 6%; use of in vitro SCE assays declined by 12%. 6. There was no change in the rate of routine use of in vivo cytogenetics or in vivo SCE assays. 7. Assays routinely performed on contract included the Salmonella assay, CHO HGPRT gene mutation, in vitro cytogenetics, in vitro UDS, in vivo cytogenetics, the micronucleus assay, L5178Y gene mutation, and the Drosophila sex-linked recessive lethal assay. 8. Four assays were being developed by five or more laboratories. These included in vitro SCE (8); the micronucleus assay (7); in vivo SCE (6); and DNA adduct formation (5). 9. A total of 17 assays had been abandoned by one or more laboratories. However, since no assay had been given up by more than three laboratories no conclusions can be drawn about the overall robustness of any of the assays on the survey form.

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This report of the Gene-Tox Assessment Panel is a compilation of data that documents the chemical testing efforts in genetic toxicology through mid-1979. It thus provides an historical perspective of the major efforts in this field and the utility of test models. The total number of chemicals tested in assays reflects chemical availability, commercial interest in specific structural types, the ease or difficulty in assay performance, as well as methodological development resulting from testing experience. Other factors that have been important in assay selection and utility are the perceptions of relevance to hazard evaluation of chemicals and the role that genetic factors may have in other disease states as well as in heritable defects. The phylogenetic diversity of test systems attests to the tremendous effort that has been applied to the testing and evaluation of the effect chemicals can have on genetic structure. The data also illustrate the fact that certain chemicals have an intrinsic capability to alter the genetic structure of cells of diverse biological origin in an heritable manner, whereas others do not. Any attempt to summarize and analyze a data base of this magnitude is a formidable task that would be almost impossible without a computer capability. A computerized system of analysis has been developed at the Environmental Mutagen Information Center (EMIC) that makes it possible to examine the performance of any particular assay in any of 30 chemical classes and to make comparisons with all the other assays individually or in designated groupings. Components of this system include: A distribution of the 2622 chemicals into 30 chemical classes with results of testing in each class. A tabulation of assay results showing the total numbers of chemicals tested, with their definitive and nondefinitive results. A subdivision of assays and results of testing into four major groups: gene mutation, chromosomal aberrations, other genotoxic effects, and in vitro cell transformation assays. These major groups are further subdivided into phylogenetic categories and type of assay. A system of analysis of results utilizing mutagenicity and carcinogenicity comparisons and phylogenetic concordance and discordance. The major utility and/or benefit of this compilation will be derived from a chemical class by chemical class comparative analysis of individual assay performance. Obviously, the data base will serve as a resource for safety evaluation of chemicals through structural correlations and biological end point analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

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The Salmonella assay has been in use for almost 15 years and can be defined as a routine test for mutagenicity and for predicting potential carcinogenicity. It detects the majority of animal carcinogens and consequently plays an important role in safety assessment. The test is also routinely used as the frontline screen for environmental samples (complex mixtures) isolated from air, water and food. This role will continue to remain an area of growth as or because sample volumes associated with these testing areas are generally very limited and more extensive testing is generally impossible. While this test, like all others, has some limitations, it is recommended that it be regularly included in all genetic testing batteries.

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Compiled results of the 1984 Genetic Toxicology Association's survey representing a total of 72 responses from governmental, contract, industrial, or academic institutions regarding the status of 32 recognized genetic toxicology assays, were as follows: Most frequently performed on a routine or occasional basis: Ames (76% of all respondents routinely or occasionally perform the assay); in vitro cytogenetics (59%); in vivo Bone Marrow Cytogenetics (56%); in vitro Sister Chromatid Exchange (56%); in vitro Unscheduled DNA Synthesis (43%); Mutation in CHO HGPRT (42%); Micronucleus test (41%); Mutation in L5178Y (36%). Assay associated with in vivo and in vitro chromosomal end points (aberrations, SCE, micronucleus) constituted the major area of increased use since the 1982 survey. Assays of developmental interest included DNAS binding both in vitro and in vivo and UDS in vivo. Quantitative analysis of the data indicated: Industrial and contract laboratories processed the largest volumes of chemicals over the broadest spectrum of assays. Industrial laboratories showed a marked increased since 1982 in testing compounds in-house. The number of assays sponsored by governmental laboratories, both in-house or subcontracted, increased from 1982. Academic institutions ranked lowest in number of compounds examined.

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The nitrosourea derivatives 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC-409962), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; NSC-79037), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (Me-CCNU; NSC-95441), and 1-methyl-1-nitrosourea (MNU; NSC-23909) were screened for mutagenic potential with Salmonella typhimurium strains G46 and TA1530 in vitro and in vivo. Alu were also mutagenic in the host-mediated assay. Four additional chemotherapeutic nitrosoureas were tested in vitro, three of which were mutagenic. The lack of activity of the fourth agent was probably a reflection of its stability in solution rather than a true indication of mutagenic potential. All eight agents were tested in a repair test with S. typhimurium strains TA1978 and TA1538. Results of this test were negative, reflecting the insensitivity of these strains to ethylating and methylating agents. The insensitivity of the host-mediated assay is also discussed.

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Fischer rat embryo cells chronically infected with Rauscher murine leukemia virus, and known to be sensitive to transformation by potent chemical carcinogens, were transformed by the weak carcinogen 4-nitropyridine-1-oxide. Transformed cells grew in semi-solid agar and produced tumors in newborn Fischer rats. Transformation was inhibited by antisera specific for the ecotropic Rauscher murine leukemia virus, but not by antisera of equal toxicity specific for xenotropic Swiss mouse AT-124 virus.

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