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)     

Why pesticides with mutagenic,carcinogenic and reproductive risks are registered in Brazil

Brazil is the biggest market for pesticides in the world. In the registration process, a pesticide must be authorized by the Institute of the Environment, Health Surveillance Agency and Ministry of Agriculture. Evaluations follow a package of toxicological studies submitted by the companies and also based on the Brazilian law regarding pesticides. We confronted data produced by private laboratories, submitted to the Institute of the Environment for registration, with data obtained from scientific databases, corresponding to mutagenicity, carcinogenicity and teratogenicity of pesticides. All studies submitted by the companies were carried out by private laboratories. From 247 pesticide formulations analyzed, none showed positive results for mutagenicity, carcinogenicity or teratogenicity. From 574 articles in the scientific literature, 84% published by public laboratories showed positive results, while 79% of those showing negative results came from private laboratories. There is an ethical concern about a conflict of interest between public/independent laboratories and private laboratories that produce data for registering pesticides. We demonstrated that there is a clear contradiction between public and private laboratories. Brazilian regulatory authorities have approved the registration of pesticides based almost exclusively on the monographs provided by the pesticide industry, because the use of scientific articles or information from the independent literature is strongly belittled by the industry. Pesticide companies argue that scientific articles cannot be trusted. Also, according to the industry, pesticide registration cannot be refused based on results from scientific articles. Thus, the registration of pesticides with mutagenic, carcinogenic and teratogenic risks has been approved in Brazil.     

Approaches to genetic control for the use of pesticides

Analysis of data available in literature has shown that 65% of 400 pesticides studied for their mutagenicity exert a mutagenic effect on any test-object. The number of revealed mutagens approaches 100% when four or more test-objects are used. Recommendations for quantitative estimation of genetic risk worked out under conditions of model experiments with standard mutagens are not available for pesticides since they are slight mutagens. The necessity for genetic stage-by-stage monitoring of pesticide use is substantiated. This monitoring should be carried out at the stage of experimental studies by means of classifications by the degree of potential mutagenic danger (the method is described) and at the stage of ecological and genetic investigations--by means of the regulation for application with due regard for the summary mutagenic background.     

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.     

Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP.

An analysis is presented in which are evaluated correlations among chemical structure, mutagenicity to Salmonella, and carcinogenicity to rats and mice among 301 chemicals tested by the U.S. NTP. Overall, there was a high correlation between structural alerts to DNA reactivity and mutagenicity, but the correlation of either property with carcinogenicity was low. If rodent carcinogenicity is regarded as a singular property of chemicals, then neither structural alerts nor mutagenicity to Salmonella are effective in its prediction. Given this, the database was fragmented and new correlations sought between the derived sub-groups. First, the 301 chemicals were segregated into six broad chemical groupings. Second, the rodent cancer data were partially segregated by target tissue. Using the previously assigned structural alerts to DNA reactivity (electrophilicity), the chemicals were split into 154 alerting chemicals and 147 non-alerting chemicals. The alerting chemicals were split into three chemical groups; aromatic amino/nitro-types, alkylating agents and miscellaneous structurally-alerting groups. The non-alerting chemicals were subjectively split into three broad categories; non-alerting, non-alerting containing a non-reactive halogen group, and non-alerting chemical with minor concerns about a possible structural alert. The tumor data for all 301 chemicals are re-presented according to these six chemical groupings. The most significant findings to emerge from comparisons among these six groups of chemicals were as follows: (a) Most of the rodent carcinogens, including most of the 2-species and/or multiple site carcinogens, were among the structurally alerting chemicals. (b) Most of the structurally alerting chemicals were mutagenic; 84% of the carcinogens and 66% of the non-carcinogens. 100% of the 33 aromatic amino/nitro-type 2-species carcinogens were mutagenic. Thus, for structurally alerting chemicals, the Salmonella assay showed high sensitivity and low specificity (0.84 and 0.33, respectively). (c) Among the 147 non-alerting chemicals less than 5% were mutagenic, whether they were carcinogens or non-carcinogens (sensitivity 0.04).     

Structural determinants of developmental toxicity in hamsters.

A CASE/MULTICASE structure activity relationship (SAR) model of developmental toxicity of chemicals in hamsters (HaDT) was developed. The model exhibited a predictive performance of 74%. The model's overall predictivity and informational content were similar to those of an SAR model of mutagenicity in Salmonella. However, unlike the Salmonella mutagenicity model, the HaDT model did not identify overtly chemically reactive moieties as associated with activity. Moreover, examination of the number and nature of significant structural determinants suggested that developmental toxicity in hamsters was not the result of a unique mechanism or attack on a specific molecular target. The analysis also indicated that the availability of experimental data on additional chemicals would improve the performance of the SAR model.     

Repair and mutagenesis in Escherichia coli K-12 after exposure to various alkyl-nitrosoguanidines.

The mutagenic and toxic effects of a series of N-alkyl-N'-nitro-N-nitrosoguanidines were examined in Escherichia coli K-12. The role of nucleotide excision repair, the SOS response, and the adaptive response in both the reduction and the production of the biological effects of these chemicals was tested. The effects of ethyl-nitrosoguanidine are similar in nucleotide excision repair-proficient and -deficient strains, but both the mutagenicity and the toxicity of alkyl groups larger than two carbons are significantly reduced by the presence of this repair system. Similarly, when alkyl groups are larger than two carbons, the umuC gene product is essential for the production of a fraction of the mutations that these lesions produce. The induction of the adaptive response had a significant effect on the toxicity of all of the chemicals tested, but its effect on mutagenicity was less uniform, having a larger effect on ethylating and propylating agents than on butylating and amylating agents.     

Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation

Mechanisms of toxicity continue to be important in developing rational strategies to deal with chemicals present in the environment. Understanding and predicting toxicity have also become a critical step in the process of drug development. Covalent binding of chemicals to macromolecules is one aspect of toxicity, and the principles and outcomes of the process are considered. Two examples of chemicals for which several aspects of metabolism and reactions are understood are aflatoxin B(1) and polyhalogenated olefins. Ethylene dibromide is a compound that is activated to genotoxic half-mustards by conjugation with glutathione or the DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT). The AGT reaction is unusual, in that crosslinking of the protein to DNA increases mutagenicity. One of the involved mechanisms is formation of N(7)-guanyl crosslinks and depurination to produce G-->T transversions; other reactions appear to yield the additional mutagenic events. The phenomenon of thiol conjugation to increase mutagenicity is widespread among bis-electrophiles.     

The mutagenic activity of selected compounds at the TK locus: rodent vs. human cells

The mutagenic (TFT resistance) and toxic responses of mouse lymphoma (MOLY) L5178Y cells and human lymphoblast (HULY) TK6 cells were compared for 13 chemicals. The mutagenic activities of 8 of the 13 chemicals (62%) examined in the HULY and MOLY assays are in agreement - the results being judged positive in both assays. However, a dramatic difference is observed when the two conditions of metabolic activation are considered separately; the overall concordance of 8/13 has been achieved by combining a 13/13 (100%) agreement in the absence of S9 with a 1/6 (17%) agreement in the presence of S9. In the absence of S9, the concentration ranges, lowest significant doses, and shapes of the concentration-response curves for both toxicity and mutagenicity were similar in spite of the differences in exposure times (4 h for MOLY, 20 for HULY) and expression times (2 days for MOLY, 3 days for HULY). The general agreement observed in the absence of S9 contrasted with the differences manifested in its presence. 6 compounds which were negative in the absence of S9 were tested in both the MOLY and HULY assays in the presence of S9. Of the 6 chemicals, only 1 was positive in both MOLY and HULY under the latter condition; 4 others were positive in MOLY and negative in HULY whereas 1 was positive in HULY and negative in MOLY.     

Mutagenicity and toxicity of carcinogenic and other hydrazine derivatives: correlation between toxic potency in animals and toxic potency in Salmonella typhimurium TA1538.

Eleven hydrazine derivatives and an aromatic amine were examined for mutagenicity and toxicity to Salmonella typhimurium. Phenylhydrazine, 2-nitrophenylhydrazine, 4-nitrophenylhydrazine, 2,4-dinitrophenylhydrazine, p-tolylhydrazine, and 4-nitroaniline were found to be frameshift mutagens (strain TA1538). Benzylhydrazine, m-hydroxybenzylhydrazine, p-hydrazinobenzoic acid, L-tyrosine hydrazide, p-aminobenzoyl hydrazide, and isoniazid were not mutagenic. All chemicals were toxic to strain TA1538. A qualitative correlation was found between the pK of the compounds and their mutagenicity. Relative toxicities of hydrazines to bacteria were found to be closely correlated with the relative toxicities of the same compounds in animals. Described herein is a methodology for the rapid prescreening of chemicals which may be used as drugs for those with a high benefit/risk ratio.     

Study of the causes of direct-acting mutagenicity in coffee and tea using the Ara test in Salmonella typhimurium

The mutagenic activities of 6 of the chemicals identified in coffee solutions were assayed with the Salmonella Ara test, under experimental conditions optimized for coffee mutagenicity. Caffeine was the only non-mutagenic compound. Among the other 5 chemicals, hydrogen peroxide was the strongest mutagen and chlorogenic acid the weakest; methylglyoxal, glyoxal and caffeic acid exhibited intermediate mutagenicities. The minimal mutagenic doses of these components correlated negatively with their relative concentrations in coffee. It was concluded that chlorogenic acid, caffeic acid, glyoxal and methylglyoxal cannot contribute alone to the mutagenicity of coffee in the Ara test, since their minimal mutagenic concentrations were much higher than their respective levels in the coffee samples assayed. By contrast, 40-60% of the mutagenic activity in coffee and also in tea could be attributed to their H2O2 contents. Catalase abolished more than 95% of the mutagenic activity of coffee, as detected by the Ara test. A similar sensitivity to catalase has been reported by other authors in relation to the coffee mutagenicity identified by the Salmonella His test. Nevertheless, the results presented in this paper suggest that the Ara forward and the His reverse mutation tests are sensitive to the mutagenicity of different constituents in coffee solutions. We propose that the His test, sensitive at high coffee doses, mainly recognizes the mutagenicity of methylglyoxal, whilst the Ara test, sensitive at low coffee doses, mainly detects the mutagenic activity of hydrogen peroxide. The data reported also suggest that the direct-acting mutagenicity(ies) detected by the Ara test in tea solutions is (are) based on similar, if not identical, mechanisms.     

A survey of EPA/OPP and open literature on selected pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim

The known aneuploidogens, benomyl and its metabolite, carbendazim (methyl 2-benzimidazole carbamate (MBC)), were selected for the third in a series of ongoing projects with selected pesticides. Mutagenicity and carcinogenicity data submitted to the US Environmental Protection Agency's (US EPA's) Office of Pesticide Programs (OPP) as part of the registration process are examined along with data from the open literature. Mutagenicity and carcinogenicity profiles are developed to provide a complete overview and to determine whether an association can be made between benomyl- and MBC-induced mouse liver tumors and aneuploidy. Since aneuploidogens are considered to indirectly affect DNA, the framework adopted by the Agency for evaluating any mode of action (MOA) for carcinogenesis is applied to the benomyl/MBC data.Both agents displayed consistent, positive results for aneuploidy induction but mostly negative results for gene mutations. Non-linear dose responses were seen both in vitro and in vivo for aneuploidy endpoints. No evidence was found suggesting that an alternative MOA other than aneuploidy may be operative. The data show that by 14 days of benomyl treatment, events associated with liver toxicity appear to set in motion the sequence of actions that leads to neoplasms. Genetic changes (as indicated by spindle impairment leading to missegregation of chromosomes, micronucleus induction and subsequent aneuploidy in bone marrow cells) can commence within 1-24h after dosing, well within the time frame for early key events. Critical steps associated with frank tumor formation in the mouse liver include hepatotoxicity, increased liver weights, cell proliferation, hypertrophy, and other steps involving hepatocellular alteration and eventual progression to neoplasms. The analysis, however, reveals weaknesses in the data base for both agents (i.e. no studies on mouse tubulin binding, no in vivo assays of aneuploidy on the target tissue (liver), and no clear data on cell proliferation relative to dose response and time dependency). The deficiencies in defining the MOA for benomyl/MBC introduce uncertainties into the analysis; consequently, benomyl/MBC induction of aneuploidy cannot be definitively linked to mouse liver carcinogenicity at this time.     

The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: the National Toxicology Program Data Base

A portion of the U.S. National Toxicology Program (NTP) Salmonella typhimurium mutagenicity data base was analyzed by CASE, an artificial intelligence SAR system. CASE identified 13 structural determinants which, with a high probability (p less than or equal to 0.05) predicted the likelihood of mutagenicity of the 243 chemicals in the data base (sensitivity = 0.989; specificity = 0.950) as well as of chemicals not included in the data base. CASE also identified an additional set of structures which were highly predictive of mutagenic potency (sensitivity = 0.949; specificity = 1.00). Even though there is little overlap among the chemicals included in the NTP and Gene-Tox Salmonella data bases, CASE found significant similarities between the structural determinants of the mutagenicity in the two data bases, thereby validating the analyses and indicating a commonality in the structural basis of mutagenicity.     

Mutagens that are not carcinogens: faulty theory or faulty tests?

In the National Toxicology Program database of 172 chemicals that were judged non-carcinogenic or equivocal in 2 year rodent studies in both sexes of rats and mice, there are 38 chemicals that were mutagenic in Salmonella. All but two of the chemicals had structural alerts for mutagenicity. The largest proportion of the mutagenic non-carcinogens were benzeneamines and substituted benzeneamines. In all, 12 of the mutagenic non-carcinogens had mutagenic carcinogen analogues, and for two chemicals, the carcinogenic analogues were not mutagenic. Non-carcinogens that were mutagenic in Salmonella also tended to be mutagenic and clastogenic in mammalian in vitro tests. The mutagenic responses are discussed and explanations offered for the mutagenicity and lack of carcinogenic activity of these chemicals.     

Effect of DNA repair inhibitors on the induction and repair of bleomycin-induced chromosome damage.

The current status of the L5178Y/TK^+/-→TK^-/- mouse-lymphona mutagenicity assay is described. Dose-survival-mutagenic response data are shown for 43 chemicals. Mutagenicity and cytotoxicity in the presence or absence of non-induced and/or Aroclor-induced rat-liver S-9 are compared for most of these chemicals. 25 of these for which usable carcinogenicity data exist have been used to construct an approximately linear relationship between oncogenic potency in vivo and mutagenic potency in this system in vitro; linearity between these two endpoints extends over a greater than 100 000-fold range in potencies. Several carcinogens which are negative or difficult to detect in the standard Ames assay are mutagenic in this mammalian cell system. These include natulan, sodium saccharin (lot S-1022), p,p′-DDE (a metabolite of DDT), dimethylnitrosamine, diethylnitrosamine and diethylstilbestrol.     

A critical evaluation of the 2011 ECHA reports on compliance with the REACH and CLP regulations and on the use of alternatives to testing on animals for compliance with the REACH regulation

On 30 June 2011, the European Chemicals Agency published two reports, one on the functioning of the REACH system, the other on the use of alternatives to animal testing in compliance with that system. The data presented are based on information gained during the first registration period under the REACH system, which included high production volume chemicals and substances of very high concern, which have the most extensive information requirements. A total of 25,460 registration dossiers were received, covering 3,400 existing, so-called 'phase-in', substances, and 900 new, so-called 'non-phase-in', substances. Data sharing and the joint submission of data are reported to have worked successfully. In the registration dossiers for these substances, results from new animal tests were included for less than 1% of all the endpoints; testing proposals (required for 'higher-tier' information requirements) were submitted for 711 in vivo tests involving vertebrate animals. The registrants mainly used old, existing experimental data, or options for the adaptation (waiving) of information requirements, before collecting new information. For predicting substance toxicity, 'read-across' was the second most-used approach, followed by 'weight-of-evidence'. In vitro toxicity tests played a minor role, and were only used when the respective test methods had gained the status of regulatory acceptance. All in all, a successful start to the REACH programme was reported, particularly since, in contrast to most predictions, it did not contribute to a significant increase in toxicity testing in animals.     

Mutagenic activity of the pesticide hexachlorobutadiene

Pesticide hexachlorobutadiene (HCBD) has been studied for its mutagenic activity in two test systems: in vivo and in vitro. The above pesticide manifested a mutagenic activity in the bone marrow cells under peroral and inhalation effect. No clastogenic action in the human peripheral lymphocyte culture was observed. Results of this study and data available in literature permit concluding that HCBD is an indirect mutagen.     

Design and analysis of experiments on mutagenicity. II. Assays involving microorganisms.

The design and statistical analysis of mutagenicity experiments involving microorganisms and a single dose of mutagen are discussed. Test statistics are derived for use in determining the mutagenicity of a chemical when survival data are available and also when such data are not available. One's likelihood (power) of correctly concluding a chemical is mutagenic is examined, and minimum total sample sizes required for 95% power are presented. It is found that one generally has greater power when survival data are available. Required precision is estimating survival is discussed in reference to type-1 and type-2 errors. The proper use of the formulae and figures presented is illustrated by examples.     

Genotoxicity of pesticides: a review of human biomonitoring studies

Pesticides constitute a heterogeneous category of chemicals specifically designed for the control of pests, weeds or plant diseases. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemical ingredients possess mutagenic properties inducing mutations, chromosomal alterations or DNA damage. Biological monitoring provides a useful tool to estimate the genetic risk deriving from an integrated exposure to a complex mixture of chemicals. Studies available in scientific literature have essentially focused on cytogenetic end-points to evaluate the potential genotoxicity of pesticides in occupationally exposed populations, including pesticide manufacturing workers, pesticide applicators, floriculturists and farm workers. A positive association between occupational exposure to complex pesticide mixtures and the presence of chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) has been detected in the majority of the studies, although a number of these failed to detect cytogenetic damage. Conflicting results from cytogenetic studies reflect the heterogeneity of the groups studied with regard to chemicals used and exposure conditions. Genetic damage associated with pesticides occurs in human populations subject to high exposure levels due to intensive use, misuse or failure of control measures. The majority of studies on cytogenetic biomarkers in pesticide-exposed workers have indicated some dose-dependent effects, with increasing duration or intensity of exposure.Chromosomal damage induced by pesticides appears to have been transient in acute or discontinuous exposure, but cumulative in continuous exposure to complex agrochemical mixtures.Data available at present on the effect of genetic polymorphism on susceptibility to pesticides does not allow any conclusion.     

An illusion of hormesis in the Ames test: statistical significance is not equivalent to biological significance

A recent report (Calabrese et al., Mutat. Res. 726 (2011) 91-97) concluded that an analysis of Ames test mutagenicity data provides evidence of hormesis in mutagenicity dose-response relationships. An examination of the data used in this study and the conclusions regarding hormesis reveal a number of concerns regarding the analyses and possible misinterpretations of the Salmonella data. The claim of hormesis is based on test data from the National Toxicology Program using Salmonella strain TA100. Approximately half of the chemicals regarded as hormetic, and the majority of the specific dose-responses identified as hormetic, were actually nonmutagenic. We conclude that the data provide no evidence of hormetic effects. The Ames test is an excellent measure of bacterial mutagenicity, but the numbers of revertant (mutant) colonies on the plate are the result of a complex interaction between mutagenicity and toxicity, which renders the test inappropriate for demonstrating hormesis in bacterial mutagenicity experiments.