Mutagenic effect of epichlorohydrin. I. Testing on human lymphocytes in vitro in comparison with TEPA.

The mutagenic effect of the monofunctional alkylating agent epichlorohydrin was tested on human lymphocytes in vitro and compared with the mutagenic effect of the polyfunctional alkylating agent TEPA. The same descending concentrations were used for both mutagens: 10(-4), 10(-5), 10(-6), 10(-7), 10(-8), 10(-9), 10(-10) and 10(-11) M. Similar types of chromosomal aberration were found, but the effect of ECHH was 4-5 times lower than that of TEPA. ECHH was found to be a mild mutagen. Different timing of mutagen application was used in the course of 56 h of cultivation of lymphocytes: 1 h before cultivation, one hour between the 24th and 25th h of cultivation and 24 h before the end of cultivation. From the results presented we conclude that the application of the chemical for the last 24 h of human lymphocyte cultivation should be recommended for routine mutagenicity testing.     

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

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)     

Assessment of the potential germ cell mutagenicity of industrial and plant protection chemicals as part of an integrated study of genotoxicity in vitro and in vivo

An approach is described that enables the germ cell mutagenicity of chemicals to be assessed as part of an integrated assessment of genotoxic potential. It is recommended, first, that the genotoxicity of a chemical be defined by appropriate studies in vitro. This should involve use of the Salmonella mutation assay and an assay for the induction of chromosomal aberrations, but supplementary assays may be indicated in specific instances. If negative results are obtained from these 2 tests there is no need for the conduct of additional tests. Agents considered to be genotoxic in vitro should then be assessed for genotoxicity to rodents. This will usually involve the conduct of a bone marrow cytogenetic assay, and in the case of negative results, a genotoxicity test in an independent tissue. Agents found to be non-genotoxic in vivo are regarded as having no potential for germ cell mutagenicity. Agents found to be genotoxic in vivo may either be assumed to have potential as germ cell mutagens, or their status in this respect may be defined by appropriate germ cell mutagenicity studies. The basis of the approach, which is supported by the available experimental data, is that germ cell mutagens will be evident as somatic cell genotoxins in vivo, and that these will be detected as genotoxins in vitro given appropriate experimentation. The conduct of appropriate and adequate studies is suggested to be of more value than the conduct of a rigid set of prescribed tests.     

Low efficiency of short-term tests in the assessment of the potential mutagenic hazard of chemical compounds to mammals

A new method of the efficiency assessment of testing mutagenicity chemical pollutants is proposed. The method is based on the selective information criterion and allows one to compare the prognostic significance of results obtained in both individual tests and test batteries. The efficiency of mutagen detection in mammals was estimated in Ames' test, the in vivo test for cytogenetic abnormalities in rodent bone-marrow cells, and the battery combining both these tests. The level of evidence for mutagenicity was determined for chemicals analyzed in these tests. Based on information obtained during the trials, a low efficiency of the analyzed tests and their battery was inferred.     

Low Efficiency of Short-Term Tests in the Assessment of the Potential Mutagenic Hazard of Chemical Compounds to Mammals

A new method of the efficiency assessment of testing mutagenicity chemical pollutants is proposed. The method is based on the selective information criterion and allows one to compare the prognostic significance of results obtained in both individual tests and test batteries. The efficiency of mutagen detection in mammals was estimated in Ames' test, the in vivo test for cytogenetic abnormalities in rodent bone-marrow cells, and the battery combining both these tests. The level of evidence for mutagenicity was determined for chemicals analyzed in these tests. Based on information obtained during the trials, a low efficiency of the analyzed tests and their battery was inferred.     

The genetic toxicity of human carcinogens and its implications

23 chemicals and chemical combinations have been designated by the International Agency for Research on Cancer (IARC) as causally associated with cancer in humans. The literature was searched for reports of their activity in the Salmonella mutagenicity assay and for evidence of their ability to induce chromosome aberrations or micronuclei in the bone marrow of mice or rats. In addition, the chemical structures of these carcinogens were assessed for the presence of electrophilic substituents that might be associated with their mutagenicity and carcinogenicity. The purpose of this study was to determine which human carcinogens exhibit genetic toxicity in vitro and in vivo and to what extent they can be detected using these two widely employed short-term tests for genetic toxicity. The results of this study revealed 20 of the 23 carcinogens to be active in one or both short-term tests. Treosulphan, for which short-term test results are not available, is predicted to be active based on its structure. The remaining two agents, asbestos and conjugated estrogens, are not mutagenic to Salmonella; asbestos is not likely to induce cytogenetic effects in the bone marrow and the potential activity of conjugated estrogens in the bone marrow is difficult to anticipate. These findings show that genetic toxicity is characteristic of the majority of IARC Group 1 human carcinogens. If these chemicals are considered representative of human carcinogens, then two short-term tests may serve as an effective primary screen for chemicals that present a carcinogenic hazard to humans.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

A combined testing protocol approach for mutagenicity testing.

The antischistosomal agent, hycanthone methanesulfonate (HMS), was employed to illustrate the utility of carrying out several mutagenicity tests in a single concurrent animal experiment. Several commonly used procedures that were successfully integrated into a multiple testing protocol included (1) metaphase analysis in bone marrow, (2) micronucleus test in bone marrow, (3) analysis of the urine for mutagenic constituents, and (4) the host-mediated assay using Salmonella typhimurium. In addition to these animal studies, in vitro mutagenicity testing with and without activation was carried out using S. typhimurium. HMS produced positive, dose--response effects in in vitro tests, metaphase analysis, micronucleus test, and urine analysis, but not in the host-mediated assay. The results of these integrated techniques suggest that such a protocol may be a benefit to those concerned with mutagenicity testing of chemicals.     

Efficiency of Batteries of Tests for Estimating Potential Mutagenicity of Chemicals

A new method for assessing the efficiency of batteries of arbitrary numbers of tests is proposed. The posterior probability of the mutagenicity of the substances studied has been estimated using discriminant analysis. The results of tests in each test system has been presented as the probability to obtain a positive result in the given test system. This has made it possible to decrease the sample size as the number of tests in the battery increased. As a result, prognostic power may be assessed even if the matrix of results is incomplete. This approach has been used to estimate the weights of evidence for mutagenic activities of 105 chemical compounds studied by means of a battery of four tests: Ames's test, the test for chromosome aberrations in vitro, the test for cytogenetic defects in vivo, and the test for dominant lethal mutations in rodents.     

Effectiveness of a battery of tests to assess the potential mutagenic danger of chemical compounds

A new method for assessing the efficiency of batteries of arbitrary numbers of tests is proposed. The posterior probability of the mutagenicity of the substances studied has been estimated using discriminant analysis. The results of tests in each test system has been presented as the probability to obtain a positive result in the given test system. This has made it possible to decrease the sample size as the number of tests in the battery increased. As a result, prognostic power may be assessed even if the matrix of results is incomplete. This approach has been used to estimate the weights of evidence for mutagenic activities of 105 chemical compounds studied by means of a battery of four tests: Ames's test, the test for chromosome aberrations in vitro, the test for cytogenetic defects in vivo, and the test for dominant lethal mutations in rodents.     

Strategies and testing methods for identifying mutagenic risks

The evolution of testing strategies and methods for identification of mutagenic agents is discussed, beginning with the concern over potential health and population effects of chemical mutagens in the late 1940s that led to the development of regulatory guidelines for mutagenicity testing in the 1970s and 1980s. Efforts to achieve international harmonization of mutagenicity testing guidelines are summarized, and current issues and needs in the field are discussed, including the need for quantitative methods of mutagenic risk assessment, dose-response thresholds, indirect mechanisms of mutagenicity, and the predictivity of mutagenicity assays for carcinogenicity in vivo. Speculation is offered about the future of mutagenicity testing, including possible near-term changes in standard test batteries and the longer-term roles of expression profiling of damage-response genes, in vivo mutagenicity testing methods, and models that better account for differences in metabolism between humans and laboratory model systems.     

Experience with mutagenicity testing of new drugs: viewpoint of a regulatory agency

Quality and quantity of mutagenicity testing were analyzed for drugs with new active compounds which were submitted for registration in the Federal Republic of Germany from mid 1982 to mid 1986. A large variety of deficiencies was found, applying to selection and number of mutagenicity tests as well as to test performances. Only 65 out of the 144 drugs submitted for registration were tested sufficiently in the initial phase of registration. From 1982 to 1986 this situation has not been changed markedly. Inadequate test performance still remains the main reason for insufficient testing, leading in some cases to artificially positive results. For in vivo tests the selection of test species was mainly motivated by technical reasons and not by characteristics of the test compound. Most of the insufficiencies were eliminated during the second phase of registration. In some cases insufficient mutagenicity testing led to consequences concerning risk-benefit assessment of the drug and its regulation.     

Genetic toxicology: can we design predictive in vivo assays?

The present analysis examines the assumptions in, the perceptions and predictivity of and the need for short-term tests (STTs) for genotoxicity in light of recent findings that most noncarcinogens from the National Toxicology Program are genotoxic (i.e., positive in one or more in vitro STTs). Reasonable assumptions about the prevalence for carcinogens (1-10% of all chemicals), the sensitivity of these STTs (ca. 90% of all carcinogens are genotoxic) and their estimated "false positive" incidence (60-75%) imply that the majority of chemicals elicit genotoxic responses and, consequently, that most in vitro genotoxins are likely to be noncarcinogenic. Thus, either the usual treatment conditions used in these in vitro STTS are producing a large proportion of artifactual and meaningless positive results or else in vitro mutagenicity is too common a property of chemicals to serve as a useful predictor of carcinogenicity or other human risk. In contrast, the limited data base on in vivo STTs suggests that the current versions of these assays may have low sensitivity which appears unlikely to improve without dropping either their 'short-term' aspect or the rodent carcinogenicity benchmark. It is suggested that in vivo genotoxicity protocols be modified to take into consideration both the fundamentals of toxicology as well as the lessons learned from in vitro genetic toxicology. In the meantime, while in vivo assays are undergoing rigorous validation, genetic toxicology, as currently practiced, should not be a formal aspect of chemical or drug development on the grounds that it is incapable of providing realistic and reliable information on human risk. It is urged that data generated in new, unvalidated in vivo genotoxicity assays be exempted from the normal regulatory reporting requirements in order to encourage industry to participate in the laborious and expensive development of this next phase of genetic toxicology.     

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.     

Methods for analysis of the mutagenicity of indirect mutagens/carcinogens in eukaryotic cells

Summary The review discusses the variety of methods for activation of indirect mutagens/carcinogens and testing them in cell cultures, especially in mammalian cell cultures.After the necessity for including metabolizing components in mutagenicity tests has been pointed out, the enzymes that transform foreign compounds metabolically, and the factors influencing them, are described. In the main section the various methods of activating indirect mutagens/carcinogens are presented. The methods of including in vivo metabolism in mutagenicity tests are: Analysis of cells from organisms contaminated with a chemical (III.1.a); body fluid-mediated mutagenesis (III.1.b); host-mediated assay (III.1.c).The following activation systems are suitable for including in vitro metabolism of test compounds in mutagenicity tests: Liver and lung perfusion (III.2.a.); organ slices and homogenates (III.2.a.); subcellular fractions (III.2.a.); cultivated cells (cell-mediated mutagenesis) (III.2.b); nonenzymatic activation systems (III.2.c).Finally the main factors that influence the metabolism of test substances are summarized. Two figures illustrate the mutagenicity tests with regard to the metabolism of mammalian livers and the methods of performing mutagenicity tests in man.     

Pentachlorophenol

Pentachlorophenol (PCP) is a substance whose widespread use, mainly in wood protection and pulp and paper mills, has led to a substantial environmental contamination. This in turn accounts for a significant exposure of the general human population, with rather high exposure levels being attained in occupational settings.Investigations on the genotoxic activity of PCP have given rise to divergent results which would seem to make an evaluation difficult. By grouping them into 3 categories a somewhat clearer picture, allowing finally an (admittedly tentative) assessment, can be obtained.PCP does seem to be at most a weak inducer of DNA damage: it produces neither DNA-strand breaks nor clear differential toxicity to bacteria in rec-assays in the absence of metabolic activation. Also in SCE induction no increase can be observed in vivo, while PCP is found marginally active in a single in vitro experiment. Metabolic activation, however, leads to prophage induction and to DNA strand breaks in. human lymphocytes, presumably through the formation of oxygen radicals. A possible further exception in this area might be the positive results in the yeast recombination tests, although their inadequate reporting makes a full evaluation difficult.PCP does not seem to induce gene (point) mutations, as most bacterial assays, the Drosophila sex-linked recessive lethal test and in vitro assays with mammalian cells did not demonstrate any effects. Marginally positive results were obtained in the mammalian spot test in vivo and in one bacterial test; the positive result in the yeast assay for cycloheximide resistance is fraught somewhat with its questionable genetic basis.PCP does, however, induce chromosomal aberrations in mammalian cells in vitro and in lymphocytes of exposed persons in vivo. Those in vivo results that were unable to provide evidence of chromosomal damage are hampered either by methodological inadequacies or by too low exposure levels.The (rodent) metabolite tetrachlorohydroquinone might be a real genotoxic agent, capable of binding to DNA and producing DNA strand breaks; this activity is probably due to semiquinone radical formation and partly mediated through active oxygen species. Since this compound has not been tested in the common bacterial and mammalian mutagenicity assays, the few ancillary results on this substance cannot be used in a meaningful human risk assessment of PCP. Furthermore, this metabolite has only been produced by human liver microsomes in vitro, but has not been detected in exposed humans in vivo. Its formation in mutagenicity test systems and its activity involving radicals might, however, help to explain some of the divergencies in the genotoxicity results.The review concludes that PCP is a weak human clastogen which may lack other genotoxic properties, although it may add somewhat to the normal oxidative damage.     

Correlation of human lymphocyte SCE frequency with smoking history

The relationship between the level of occupational exposure to epichlorohydrin (ECHH) and the clastogenic effect was studied on a group of 33 workers. The effect of ECHH was assessed by differences in the frequency of chromosome aberrations in peripheral blood lymphocytes in ECHH-exposed and control groups. In the group exposed to the average ECHH concentration, 0.384 mg X m-3, during the last 6 months, the cytogenetic analysis revealed 2.00 +/- 0.23% AC (aberrant cells) (0.0203 B/C, breaks per cell) as compared with 1.68 +/- 0.23% AC (0.0172 B/C) in the matching controls. These results indicate that an average concentration lower than 0.40 mg X m-3 ECHH in the working atmosphere has no significant clastogenic effect on human peripheral lymphocytes.     

The in vivo and in vitro genotoxicity of aromatic amines in relationship to the genotoxicity of benzidine.

Benzidine and 12 related aromatic amines have been studied for the effects of substituent groups and pi orbital conjugation on their genotoxicity as measured by their mutagenicity in vitro with Salmonella and by chromosomal aberrations (CA) in vivo in the bone-marrow cells of mice. The in vitro studies indicated increases in mutagenicity with increases in the electron withdrawing ability of para' substituents. Mutagenicity also increases with increased conjugation as shown by the degree of planarity of the biphenyl compounds and by comparing the mutagenicities of biphenyl amines to stilbenes as well as to ethylene bridged diphenyl compounds. The relative in vitro mutagenicity results were not predictive of relative in vivo CA results. The 3 most genotoxic compounds in vivo were the conjugated amines without substituents in the para' position. The CA values for 4-aminostilbene were exceptionally high. These in vivo results indicate increased genotoxicity for benzidine analogs without substitution in the para' position.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.