Induction of tunica vaginalis mesotheliomas in rats by xenobiotics.

To better understand the relevance of tunica vaginalis mesotheliomas (TVM) to human cancer risk, we examined the nature of TVM responses in 21 published rat cancer bioassays against the backdrop of the biology and molecular biology of mesothelium, and of spontaneous and treatment-induced TVM. Although relatively rare in all species including humans, TVM are seen most frequently in F344 male rats, as opposed to other rat strains, and are causally associated with the high background incidence of Leydig-cell tumors of the testes of these rats. Hormone imbalance brought about by perturbations of the endocrine system is proposed as a key factor leading to both spontaneous and treatment-associated TVM. Of 21 F344 rat studies with a treatment-associated TVM response, 7 were judged to have a nonsignificant to marginal response, 11 had a robust TVM response, and 3 were noninformative due to early mortality from other induced tumors. Of the 11 chemicals with robust responses, 8 were directly mutagenic in Salmonella and 3 are known to be mutagenic after metabolism. Only 2 of the 7 with nonsignificant to marginal responses were Ames test positive. TVM induction is a male F344 rat-specific event, and chemicals/agents that induce only TVM in the male F344 rat from a typical two-sex rat and mouse chronic bioassay are likely irrelevant in human risk assessment.

Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing.

This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.

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.

HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei.

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of "normal" values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin-B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5 per thousand and the interquartile range was between 3 and 12 per thousand. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14-24%). Statistical analyses were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.

The genetic toxicity of 3,3',4,4'-tetrachloroazobenzene and 3,3',4, 4'-tetrachloroazoxybenzene: discordance between acute mouse bone marrow and subchronic mouse peripheral blood micronucleus test results.

3,3',4,4'-Tetrachloroazobenzene (TCAB) and 3,3',4, 4'-tetrachloroazoxybenzene (TCAOB) are dioxin-like chemicals that were investigated for toxicity in 13-week gavage studies in male and female B6C3F(1) mice and F344N rats by the National Toxicology Program. As part of the comprehensive toxicological investigation of these chemicals, peripheral blood smears from mice treated 5 days per week for 13 weeks with 0.1-30mg/kg/day TCAB or TCAOB were analyzed for the frequency of micronucleated (MN) normochromatic erythrocytes (NCE). Both chemicals produced significant increases in MN-NCE in male and female mice. In contrast to these positive results in subchronic exposure studies, no significant increases were seen in acute bone marrow MN tests in male mice administered three daily injections of 50-200mg/kg/day TCAB and TCAOB. The results with TCAB and TCAOB suggest that the routine integration of MN tests with subchronic toxicity studies may allow detection of mutagenic activity for some chemicals that fail to elicit responses in short-term, high dose tests. In addition, the integration of mutagenicity tests into general toxicity tests reduces the use of laboratory animals and the cost of the testing.

The proportions of mutagens among chemicals in commerce.

It has been estimated that there are approximately 80,000 chemicals in commerce. Thus, it is not possible to test all these substances for mutagenicity and carcinogenicity; it is possible, however, to test or make estimates from selected subsets of these chemicals. For example, in the U.S. National Toxicology Program (NTP), 35% of the chemicals tested for mutagenicity in Salmonella were positive, as were 52% of the chemicals tested for carcinogenicity in rodents. In contrast, in the U.S. EPA Gene-Tox database, the proportions of chemicals that are Salmonella mutagens is 56%. These and other databases may be biased toward positive responses because they generally have been developed to look at specific structural or use classes of chemicals or chemicals suspected of genetic or carcinogenic activity. To address the question of the proportions of mutagens among all chemicals in commerce, a database of 100 chemicals was created from a random selection of chemicals in commerce. These chemicals were tested for mutagenicity in Salmonella and 22% were mutagenic. The mutagenicity of the 46 highest U.S. production organic chemicals was also compiled; 20% were mutagenic. These values provide a more accurate estimate of the proportions of mutagens among chemicals in commerce than can be derived from published mutagenicity databases.

The HUman MicroNucleus Project--An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans.

The International Collaborative Project on Micronucleus Frequency in Human Populations (HUMN) was organized to collect data on micronucleus (MN) frequencies in different human populations and different cell types. The test procedures considered by this project are assays using human lymphocytes (cytokinesis-block method), exfoliated epithelial cells, and other cell types. Data (including descriptions of the populations monitored, detailed test protocols, and test results) are being obtained from a large number of laboratories throughout the world and are being entered into a unified database. The information will be used to: (1) determine the extent of variation of 'normal' values for different laboratories and the influence of other factors potentially affecting baseline MN frequency, e.g., age, gender and life-style; (2) provide information on the effect of experimental protocol variations on MN frequency measurements; (3) design and test optimal protocols for the different cell types; and (4) determine the extent to which MN frequency is a valid biomarker of ageing and risk for diseases such as cancer.

Predicting rodent carcinogenicity using potency measures of the in vitro sister chromatid exchange and chromosome aberration assays.

In vitro sister chromatid exchange (SCE) and chromosome aberration (ABS) tests have been extensively used to identify potential rodent carcinogens. A number of measures of potency were developed to describe in vitro SCE and ABS test results: the dose needed to induce a unit increase over the control; the lowest effective dose; the slope of the ordinary linear regression; the maximum observed slope; and the maximum fold increase over background. The ability of these potency measures to predict the qualitative and quantitative carcinogenicity of chemicals was compared to the predictivity of the qualitative in vitro responses. The results of the analyses showed that the quantitative measures of the SCE or ABS responses only minimally increased the predictivity of carcinogenesis when compared to the predictivity using the qualitative responses.

Induction of micronucleated erythrocytes in rodents by diisopropylcarbodiimide and dicyclohexylcarbodiimide: dependence on exposure protocol.

The induction of micronucleated erythrocytes by diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) was investigated as part of a U.S. National Toxicology Program (NTP) evaluation of the subchronic toxicity of these chemicals. Analysis of peripheral blood smears from male and female B6C3F1 mice exposed to 17.5-140.0 mg DIC/kg/day by skin painting for 13 weeks revealed dose-related increases in the frequency of micronucleated normochromatic erythrocytes (MN-NCE) in both sexes. Results of a similar 13-week peripheral blood micronucleus (MN) test with DCC (1.5-12.0 mg/kg/day) were also positive, although the increases in MN-NCE were not as great as those observed with DIC. In contrast to the positive results of the subchronic skin-painting studies in mice, acute bone marrow MN studies with DIC and DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Both the acute and the subchronic exposures included doses that produced clinical signs of toxicity. Acute mouse bone marrow MN tests with DIC administered in single or triple i.p. injection protocols were subsequently conducted to determine if the differing responses between mice and rats were due to species or protocol differences. The results of these acute tests were negative or equivocal. Because the subchronic studies produced positive results, it was hypothesized that these carbodiimides required multiple treatments over an extended period of time to produce an increase in MN-erythrocytes. To confirm the original response, a second dermal subchronic study was conducted with DIC; the protocol was modified to include sequential blood samplings to permit monitoring MN frequencies over time. The data demonstrated a small but consistent induction of micronucleated erythrocytes in mice treated with DIC by skin painting.

Identification of rodent carcinogens and noncarcinogens using genetic toxicity tests: premises, promises, and performance.

The basic premises that guide genetic toxicity testing for identifying carcinogens and to support administrative and regulatory decisions are: the Salmonella mutagenicity test is a necessary component of testing schemes; a chromosome aberration test is needed in addition to a gene mutation test; a mammalian cell mutagenicity test is needed in addition to the Salmonella test; in vivo tests are needed to confirm the results of in vitro tests; and test batteries are more predictive than the individual tests of the battery. Results from the Salmonella mutagenicity, in vitro chromosome aberration, mutations in mouse lymphoma cells, rodent bone marrow micronucleus, and rodent carcinogenicity tests, performed by the U.S. National Toxicology Program, were used to evaluate these premises. A positive Salmonella test was most predictive of carcinogenicity. However, the data do not support using the other tests in addition to Salmonella for predicting carcinogenicity. The genetic toxicity tests did not complement each other, and batteries or combinations of the tests were no more predictive of carcinogenicity than Salmonella alone. If a chemical is mutagenic in Salmonella it should be considered a potential rodent carcinogen, unless ancillary information suggests otherwise. Positive responses in the other in vitro or in vivo tests do not increase the probability that the chemical is a carcinogen, and negative responses in the other tests do not diminish the implications of the positive Salmonella response.

Genetic toxicity studies of 1,2,3,4-tetrahydro-9-acridinamine (tacrine).

The mutagenicity and clastogenicity of 1,2,3,4-tetrahydro-9-acridinamine (tacrine) were studied in vitro using the Salmonella mutagenicity test and the induction of chromosome aberrations in Chinese hamster ovary (CHO) cells, and in the mouse bone marrow micronucleus test in vivo. This chemical is currently being used to treat dementia arising from Alzheimer's Disease. Tacrine was mutagenic in Salmonella but did not produce chromosome damage in CHO cells or in mouse bone marrow cells. A clear mutagenic response was seen in strain TA97 with rat and hamster liver S9; inconsistent results were obtained without S9. No mutagenicity was seen in strains TA98 and TA100 without S9, and inconsistent results were seen with S9. There was no induction of chromosome aberrations in cultured CHO cells with or without S9. Oral administration to mice of tacrine daily for three days did not result in the induction of micronuclei in their bone marrow cells. The mutagenic response in Salmonella, and the structure of the molecule, suggests that tacrine may be carcinogenic when tested in rodents. This information must be considered when preparing benefit-risk determinations for medical uses of this substance.

Formation of 8-hydroxy-2'-deoxyguanosine following treatment of 2'-deoxyguanosine or DNA by hydrogen peroxide or glutathione.

We have demonstrated that free radicals generated by hydrogen peroxide (H2O2), in the presence of divalent iron (Fe2+) and a chelator (EDTA), oxidize 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG). The 8-OHdG formed by this reaction was isolated and quantitated using reverse-phase HPLC with UV and electrochemical detection. A 1-h incubation of dG with H2O2 caused a 50% increase in 8-OHdG over background, which increased to 100% after 2 h. However, when an H2O2-generating system [glutathione (GSH), Fe2+, EDTA] was used, there was no increase in 8-OHdG yield after the 1-h incubation, but up to a 50% increase over background was observed with GSH after 2-h incubation. Attempts to detect increased levels of 8-OHdG after H2O2- or GSH-treatment of purified calf thymus or rat DNA, or purified Salmonella typhimurium DNA were not successful. This may have been because the treatment procedures used generated 8-OHdG in the control samples at sufficiently high levels to mask any H2O2-induced responses that may have been present. This artifactual production of 8-OHdG has presented a problem in all in vitro studies to date. In contrast, treatment of Salmonella cells (strain TA104) with increasing concentrations of H2O2, caused a doubling in the 8-OHdG yield. GSH-treatment of strain TA104 cells under the same conditions did not result in an increase of 8-OHdG. The study presented here shows that the ubiquitous molecule H2O2 can play a major role in DNA oxidation, mutation, and damage.

Predicting rodent carcinogenicity from mutagenic potency measured in the Ames Salmonella assay.

Many in vitro tests have been developed to identify chemicals that can damage cellular DNA or cause mutations, and secondarily to identify potential carcinogens. The test receiving by far the most use and attention has been the Salmonella (SAL) mutagenesis test developed by Ames and colleagues [(1973): Proc Natl Acad Sci USA 70:2281-2285; (1975): Mutat Res 31:347-364], because of its initial promise of high qualitative (YES/NO) predictivity for cancer in rodents and, by extension, in humans. In addition to the initial reports of high qualitative predictivity, there was also an early report by Meselson and Russell [in Hiatt HH et al (1977): "Origins of Human Cancer, Book C: Human Risk Assessment," pp 1473-1481] of a quantitative relationship between mutagenic potency measured in SAL and carcinogenic potency measured in rodents, for a small number of chemicals. However, other reports using larger numbers of chemicals have found only very weak correlations. The primary purpose of this study was to determine whether mutagenic potency, as measured in a number of different ways, could be used to improve predictivity of carcinogenicity, either qualitatively or quantitatively. To this end, eight measures of SAL mutagenic potency were used. This study firmly establishes that the predictive relationship between mutagenic potency in SAL and rodent carcinogenicity is, at best, weak. When predicting qualitative carcinogenicity, only qualitative mutagenicity is useful; none of the quantitative measures of potency considered improves the carcinogenicity prediction. In fact, when qualitative mutagenicity is forced out of the model, the quantitative measures are still not predictive of carcinogenicity. When predicting quantitative carcinogenicity, several possible methods were considered for summarizing potency over all experiments; however, in all cases, the relationship between mutagenic potency predictors and quantitative carcinogenicity is very weak.

Cytogenetic studies of sodium fluoride in mice.

The cytogenetic effects of sodium fluoride (NaF) were measured in mice following administration in the drinking water for 6 weeks. Bone fluoride levels were determined and showed a dose-related incorporation of fluoride. Micronuclei were measured in peripheral blood erythrocytes following 1 and 6 weeks of NaF administration. Bone marrow cell preparations were examined for the presence of chromosome aberrations following 6 weeks of treatment; metaphase and anaphase cells were examined. Anaphase cells were scored in three independent laboratories, two of which also scored metaphase cells from the same slides. No increases in micronuclei were seen in peripheral erythrocytes at either time point, and no increases in chromosome aberrations were seen in bone marrow cells when metaphase or anaphase cells were examined. A concurrent positive control, cyclophosphamide, produced significant increases in peripheral blood cell micronuclei and in chromosome aberrations in bone marrow cells in metaphase. No increases in aberrations were seen in the same cyclophosphamide-treated mice when anaphase cells were examined.