Genetic activity profiles and pattern recognition in test battery selection

Computer-generated genetic activity profiles and pairwise matching procedures may aid in the selection of the most appropriate short-term bioassays to be used in test batteries for the evaluation of the genotoxicity of a given chemical or group of chemicals. Selection of test batteries would be based on a quantitative comparative assessment of the past performance of similar tests applied to other chemicals of the same structural group. The information potentially available for test-battery selection through the use of this pattern-recognition technique is considerably greater than the qualitative results obtained from individual short-term tests. Application of the method should further our understanding of the relationships between chemical properties and genotoxic responses obtained in short-term bioassays and also may contribute to our knowledge of the mechanisms of complex processes such as carcinogenesis. This approach to battery selection should be augmented by careful consideration of established principles of genetic toxicity testing; that is, a chemical should be evaluated in a battery of tests representing the full range of relevant genetic endpoints.     

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.     

Animal carcinogenicity studies: 3. Alternatives to the bioassay

Conventional animal carcinogenicity tests take around three years to design, conduct and interpret. Consequently, only a tiny fraction of the thousands of industrial chemicals currently in use have been tested for carcinogenicity. Despite the costs of hundreds of millions of dollars and millions of skilled personnel hours, as well as millions of animal lives, several investigations have revealed that animal carcinogenicity data lack human specificity (i.e. the ability to identify human non-carcinogens), which severely limits the human predictivity of the bioassay. This is due to the scientific inadequacies of many carcinogenicity bioassays, and numerous serious biological obstacles, which render profoundly difficult any attempts to accurately extrapolate animal data in order to predict carcinogenic hazards to humans. Proposed modifications to the conventional bioassays have included the elimination of mice as a second species, and the use of genetically-altered or neonatal mice, decreased study durations, initiation-promotion models, the greater incorporation of toxicokinetic and toxicodynamic assessments, structure-activity relationship (computerised) systems, in vitro assays, cDNA microarrays for detecting changes in gene expression, limited human clinical trials, and epidemiological research. The potential advantages of non-animal assays when compared to bioassays include the superior human specificity of the results, substantially reduced time-frames, and greatly reduced demands on financial, personnel and animal resources. Inexplicably, however, the regulatory agencies have been frustratingly slow to adopt alternative protocols. In order to decrease the enormous cost of cancer to society, a substantial redirection of resources away from excessively slow and resource-intensive rodent bioassays, into the further development and implementation of non-animal assays, is both strongly justified and urgently required.     

The genetic toxicology of putative nongenotoxic carcinogens

This report examines a group of putative nongenotoxic carcinogens that have been cited in the published literature. Using short-term test data from the U.S. Environmental Protection Agency/International Agency for Research on Cancer genetic activity profile (EPA/IARC GAP) database we have classified these agents on the basis of their mutagenicity emphasizing three genetic endpoints: gene mutation, chromosomal aberration and aneuploidy. On the basis of results of short-term tests for these effects, we have defined criteria for evidence of mutagenicity (and nonmutagenicity) and have applied these criteria in classifying the group of putative nongenotoxic carcinogens. The results from this evaluation based on the EPA/IARC GAP database are presented along with a summary of the short-term test data for each chemical and the relevant carcinogenicity results from the NTP, Gene-Tox and IARC databases. The data clearly demonstrate that many of the putative nongenotoxic carcinogens that have been adequately tested in short-term bioassays induce gene or chromosomal mutations or aneuploidy.     

The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens

The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.     

Toxicity analysis of freshwater and marine sediments with meio- and macrobenthic organisms: a review

Benthic metazoans play a key role as test organisms in toxicity analyses of aquatic ecosystems. This report gives an overview of the species of benthic metazoans used for the assessment of toxicity in freshwater and marine sediments, as well as of the criteria relevant to the choice between test species and procedures. The main applications of these organisms are mono-species bioassays, test-batteries, analyses of benthic communities and bioaccumulation studies. Sediment toxicity assays, including acute and chronic exposures, have been developed for nematodes, insects, oligochaetes, polychaetes, crustaceans, molluscs and echinoderms. At least 30 species of freshwater and 71 species of marine and estuarine benthic metazoans have thus far been used in sediment toxicity bioassays. Although aquatic pollution is a world-wide problem, most sediment toxicity bioassays have been developed for organisms native to Europe and North America. The most common bioassay endpoints are mortality, development, growth and behavioural responses. The value of genetic, biochemical, physiological and pathological responses as toxicity endpoints is currently being investigated. The quest for additional test species and protocols is still a worthwhile endeavour in sediment ecotoxicology.     

An analysis of the Gene-Tox Carcinogen Data Base

The Gene-Tox Carcinogen Data Base is an evaluated source of cancer data on 506 chemicals selected in part for their previous assessment in genetic toxicology bioassays. This data base has been analyzed for the distribution of these chemicals into chemical classes. The major chemical classes (6% or greater of the total data base) are: acyl-, alkyl-, and aryl-halides; alcohols and phenols; aliphatic and aromatic amines, amides, and sulfonamides; benzene-ring-containing chemicals; organo-lead, -mercury, -phosphorous compounds, metals and derivatives, phosphoric acid esters, and phosphoramides; and polycyclic aromatic hydrocarbons. Cancer studies representing a subset of the Gene-Tox Carcinogen Data Base, 199 chemicals which were rated as Sufficient Positive/Negative or Limited Positive/Negative, were examined for distribution of those studies by animal species, gender, route of chemical administration, duration of study, major tumor sites, and major tumor types. These analyses revealed that the Gene-Tox Carcinogen Data Base contains a large number of lifetime studies involving the rat and mouse treated by oral routes of administration. The major organs that were targets were: liver, lung, skin, forestomach, bladder, and mammary gland, while the major tumor types were: carcinoma, sarcoma, papilloma, and adenoma. Chemicals in the data base have been assessed for species-specific carcinogenic effects, and these results indicate that for mice and rats there is a high correspondence (85%). This number is higher than that (71%) reported by Tennant et al. (1986) based on the recent results of 72 chronic cancer bioassays performed by the National Toxicology Program. This difference is probably based on the nature of the chemicals selected for inclusion in both data bases. Although the absolute value of this correspondence is unknown, it would seem to be within this range. When chemicals in the Gene-Tox Carcinogen Data Base were examined for their previous evaluation in 73 genetic toxicology bioassays, only 26 of these bioassays had 30 or more chemicals. In these 26 bioassays, the prevalence of positive chemicals was generally greater than 80-90%. This suggests that a thorough evaluation of genetic toxicology bioassays in regard to their ability to predict carcinogenic effects in animals is premature at this time.     

Prediction of non-genotoxic carcinogenesis in rats using changes in gene expression following acute dosing

Non-genotoxic carcinogenicity of chemicals is currently routinely evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for non-genotoxic carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens (NGTCs) or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR). We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 non-genotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 non-genotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected at both 1 and 5 days. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.     

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).     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo

There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus (MN) test (traditionally the most widely-used test) is negative. Not all rodent carcinogens give positive results in the micronucleus test, and so it has been common practice to include a second in vivo assay such as the unscheduled DNA synthesis (UDS) test. This has proved useful but is usually limited to analysis of rodent (usually rat) liver. With the increased evaluation and use of other in vivo assays, e.g. for transgenic mutations (TG) and DNA damage (Comet assay) it was important to investigate their usefulness. We therefore examined the published in vivo UDS, TG and Comet-assay results for 67 carcinogens that were negative or equivocal in the micronucleus test. Between 30 and 41 chemicals were evaluated in each of the three in vivo tests, with some overlap. In general, the UDS test was disappointing and gave positive results with <20% of these carcinogens, some of which induced tumours in rat liver and produced DNA adducts in vivo. The TG assay gave positive responses with >50% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus-negative or equivocal carcinogens. This pattern of results was virtually unchanged when the in vitro profile (gene mutagen or clastogen) was taken into account. High sensitivity (ability to detect carcinogens as positive) is only really useful when the specificity (ability to give negative results with non-carcinogens) is also high. Based on small numbers of publications with non-carcinogens, the TG and Comet assays gave negative results with non-carcinogens on 69 and 78% of occasions, respectively. Although further evaluation of the Comet and TG assays, particularly with non-carcinogens, is needed, these data suggest that they both should play a more prominent role in regulatory testing strategies than the UDS test.     

Implications for genetic toxicology of the chromosomal breakage syndromes

The activation of oncogenes and our knowledge of the chromosome breakage syndromes show that both intragenic mutations and chromosomal aberrations are important in carcinogenesis. Each suggests that an agent could produce genetic changes in a tissue without producing cancer there, if the types of genetic change do not match: chromosomal aberrations may be irrelevant in the mammary epithelium but be very significant in the bone marrow, and vice versa. This has vital implications for genetic toxicology: (1) both gene mutations and chromosomal aberrations should be measured, and (2) carcinogens may be mutagenic in tissues in which they are not carcinogenic. One might therefore expect in vivo assays for mutagenicity to correlate rather well with cancer bioassays; unfortunately, the bioassays themselves seem faulty. If cancer bioassays are valid, they would be reproducible. If bioassays are reproducible, they would be internally consistent. The information supplied by Tennant et al. (1987) for their validation of in vitro assays gives data from both sexes in rats and mice for 70 chemicals. When the data are analyzed site-by-site, positive results were not replicated in the other sex or in the other species much of the time: in half the cases the other sex does not give the same result; in two-thirds of the cases the other species does not give the same result. There are 3 potential explanations for these differing results: (1) genuine sex-specific carcinogens are common, (2) genuine species-specific carcinogens are common, or (3) the bioassay does not replicate well, i.e., is erratic. The third possibility best explains the data. The apparent inability of short-term in vitro tests to discriminate well between carcinogens and non-carcinogens may be more a reflection of the cancer bioassays that were used to determine which chemicals were carcinogenic than any defect in the assays. In this situation in vivo assays can scarcely be expected to do better even if they are better.     

Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics

Two year rodent bioassays play a key role in the assessment of carcinogenic potential of chemicals to humans. The seventh amendment to the European Cosmetics Directive will ban in 2013 the marketing of cosmetic and personal care products that contain ingredients that have been tested in animal models. Thus 2-year rodent bioassays will not be available for cosmetics/personal care products. Furthermore, for large testing programs like REACH, in vivo carcinogenicity testing is impractical. Alternative ways to carcinogenicity assessment are urgently required. In terms of standardization and validation, the most advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs). Although CTAs do not mimic the whole carcinogenesis process in vivo, they represent a valuable support in identifying transforming potential of chemicals. CTAs have been shown to detect genotoxic as well as non-genotoxic carcinogens and are helpful in the determination of thresholds for genotoxic and non-genotoxic carcinogens. The extensive review on CTAs by the OECD (OECD (2007) Environmental Health and Safety Publications, Series on Testing and Assessment, No. 31) and the proven within- and between-laboratories reproducibility of the SHE CTAs justifies broader use of these methods to assess carcinogenic potential of chemicals.     

The unique role of rodents in the detection of possible human carcinogens and mutagens

Some of the probable reasons underlying the observation that not all chemicals shown to be genotoxic in vitro are capable of eliciting tumours in rodents or humans are discussed using appropriate examples. It is suggested that a substantial proportion of the resources currently available for conducting rodent carcinogenicity bioassays should be employed in the short-term evaluation in vivo of some of the many hundreds of chemicals recently defined as genotoxic in vitro, rather than in the protracted evaluation of a few chemicals, often of unknown activity in vitro, for carcinogenicity. A decision tree approach to the evaluation of chemicals for human mutagenic/carcinogenic potential is presented which is at variance with the construction and philosophy of many of the current legislative guidelines. The immediate need for the adoption of one of the available short-term in vivo liver assays, and/or the development of a short-term in vivo rodent assay capable of concomitantly monitoring different genetic end-points in a range of organs or tissues is emphasized.     

Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the U.S. National Toxicology Program

This paper is an extension and update of an earlier review published in this journal (Ashby and Tennant, 1988). A summary of the rodent carcinogenicity bioassay data on a further 42 chemicals tested by the U.S. National Toxicology Program (NTP) is presented. An evaluation of each chemical for structural alerts to DNA-reactivity is also provided, together with a summary of its mutagenicity to Salmonella. The 42 chemicals were numbered and evaluated as an extension of the earlier analysis of 222 NTP chemicals. The activity patterns and conclusions derived from the earlier study remain unchanged for the larger group of 264 chemicals. Based on the extended database of 264 NTP chemicals, the sensitivity of the Salmonella assay for rodent carcinogens is 58% and the specificity for the non-carcinogens is 73%. A total of 32 chemicals were defined as equivocal for carcinogenicity and, of these, 11 (34%) are mutagenic to Salmonella. An evaluation is made of instances where predictions of carcinogenicity, based on structural alerts, disagree with the Salmonella mutagenicity result (12% of the database). The majority of the disagreements are for structural alerts on non-mutagens, and that places these alerts as a sensitive primary screen with a specificity lower than that of the Salmonella assay. That analysis indicates some need for assays complementary to the Salmonella test when screening for potential genotoxic carcinogens. It also reveals that the correlation between structural alerts and mutagenicity to Salmonella is probably greater than 90%. Chemicals predicted to show Michael-type alkylating activity (i.e., CH2 = CHX; where X = an electron-withdrawing group, e.g. acrylamide) have been confirmed as a structural alert, and the halomethanes (624 are possible) have been classified as structurally-alerting. To this end an extended carcinogen-alert model structure is presented. Among the 138 NTP carcinogens now reviewed, 45 (33%) are non-mutagenic to Salmonella and possess a chemical structure that does not alert to DNA-reactivity. These carcinogens therefore either illustrate the need for complementary genetic screening tests to the Salmonella assay, or they represent the group of non-genotoxic carcinogens referred to most specifically by Weisburger and Williams (1981); the latter concept is favoured.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles

One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Muller, 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] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are "false" requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement.     

Reevaluation of the 9 compounds reported conclusive positive in yeast Saccharomyces cerevisiae aneuploidy test systems by the Gene-Tox Program using strain D61.M of Saccharomyces cerevisiae

The state of aneuploidy test methodology was appraised by the U.S. Environmental Protection Agency in 1986 in analyzing published data. In Saccharomyces cerevisiae 9 chemicals were reported to be conclusive positive for aneuploidy induction in either mitotic or meiotic cells. We reevaluated these 9 chemicals using Saccharomyces cerevisiae D61.M, a strain that detects mitotic chromosome malsegregation. Acetone (lowest effective dose (LED): 40 microliters/ml), bavistan (LED: 5 micrograms/ml), benomyl (LED: 30 micrograms/ml) and oncodazole (LED: 4 micrograms/ml) induced a dose-dependent increase in the frequencies of chromosomal malsegregation. Ethyl methanesulfonate (EMS; highest tested dose (HTD): 1000 micrograms/ml) and methyl methanesulfonate (MMS; HTD: 100 micrograms/ml) did not induce malsegregation but were both potent inducers of other genetic events, detected by an increase in the frequencies of cyhR cells. No increases in both endpoints (malsegregation and other genetic events) were observed after treatment of S. cerevisiae D61.M with cyclophosphamide (CP; HTD: 16 mg/ml) in the absence of S9, p-D,L-fluorophenylalanine (p-FPA; HTD: 250 micrograms/ml) and phorbol-12-myristate-13-acetate (TPA; HTD: 50 micrograms/ml). A marginal increase in the frequency of mitotic chromosome malsegregation was obtained with cyclophosphamide in the presence of S9. Thus our test results largely disagree with those previously published by various authors and taken as conclusive by EPA. We interpret the discrepancies to be due to lack of properly controlled testing (e.g., no check for multiple mutational events). Only with a careful test design it is possible to discriminate between chemicals inducing only chromosome loss and no other genetic effects (e.g., acetone, oncodazole), chemicals inducing a variety of genetic damage but no chromosome loss (e.g., EMS, MMS) and chemicals inducing neither chromosome loss nor other genetic events in yeast (e.g., TPA, p-FPA).     

Animal carcinogenicity studies: 2. Obstacles to extrapolation of data to humans

Due to limited human exposure data, risk classification and the consequent regulation of exposure to potential carcinogens has conventionally relied mainly upon animal tests. However, several investigations have revealed animal carcinogenicity data to be lacking in human predictivity. To investigate the reasons for this, we surveyed 160 chemicals possessing animal but not human exposure data within the US Environmental Protection Agency chemicals database, but which had received human carcinogenicity assessments by 1 January 2004. We discovered the use of a wide variety of species, with rodents predominating, and of a wide variety of routes of administration, and that there were effects on a particularly wide variety of organ systems. The likely causes of the poor human predictivity of rodent carcinogenicity bioassays include: 1) the profound discordance of bioassay results between rodent species, strains and genders, and further, between rodents and human beings; 2) the variable, yet substantial, stresses caused by handling and restraint, and the stressful routes of administration common to carcinogenicity bioassays, and their effects on hormonal regulation, immune status and predisposition to carcinogenesis; 3) differences in rates of absorption and transport mechanisms between test routes of administration and other important human routes of exposure; 4) the considerable variability of organ systems in response to carcinogenic insults, both between and within species; and 5) the predisposition of chronic high dose bioassays toward false positive results, due to the overwhelming of physiological defences, and the unnatural elevation of cell division rates during ad libitum feeding studies. Such factors render profoundly difficult any attempts to accurately extrapolate human carcinogenic hazards from animal data.     

The non-genotoxicity to rodents of the potent rodent bladder carcinogens o-anisidine and p-cresidine.

The two potent rodent bladder carcinogens o-anisidine and p-cresidine, and the structurally related non-carcinogen 2,4-dimethoxyaniline, have been extensively evaluated for genotoxicity to rodents and found to be inactive. Most data were generated on o-anisidine, an agent that is also only marginally genotoxic in vitro. The two carcinogens induced methaemoglobinaemia in rodents indicating that the chemicals are absorbed and metabolically oxidized. Despite their total lack of genotoxicity in vivo, the two carcinogens have the hall-marks of being genotoxic carcinogens given that most test animals of both sexes of B6C3F1 mice and F344 rats are reported to have succumbed rapidly to malignant bladder cancer. No reasons for this dramatic conflict of test data are so far apparent. The experiments described involve, in one or other combination, 2 strains of mice (including B6C3F1) and 4 strains of rat (including F344), the use of oral and i.p routes of exposure and observations made after 1, 3 or 6 doses of test chemical. 6 tissues (including the rat bladder) were assayed using 3 genetic endpoints (unscheduled DNA synthesis, DNA single-strand breaks and micronuclei induction). Aroclor-induced rats were employed in one set of experiments with o-anisidine. In the case of one set of mouse bone-marrow micronucleus experiments the same batch of the 3 chemicals as used in the cancer bioassays, and the same strain of mouse, were used. Possible further experiments and the implications of these findings are discussed.