On the efficiency of age-adjusted tests in animal carcinogenicity experiments

Based on asymptotic relative efficiency calculations, Ryan (1985, Biometrics 41, 525-531) concludes that, in the analysis of animal carcinogenicity experiments, age-adjusted tests of tumor rates should be routinely preferred to simple proportions tests for both lethal and nonlethal tumors. We recalculate the asymptotic efficiencies of the simple proportions test relative to the log-rank test for the lethal tumor case. For a simplified model it is shown that the relative efficiency may be easily computed as a function of the crude tumor rate and the survival rate at the time of terminal sacrifice. More generally, we calculate by numerical quadrature the asymptotic relative efficiency for all models considered by Ryan and, using simulations, examine the relevance of asymptotic efficiencies to typical sample sizes. Contrary to the numerical results of Ryan, we find, for experiments with good survival and typical tumor rates, that the relative efficiencies are greater than 95%, usually about 99%. In the nonlethal tumor case, similar results follow from Ryan for tumor rates and survival rates typically encountered in practice. As it is often difficult to determine whether or not a tumor is lethal, we conclude for equal interim mortality rates, that the simple proportions test is usually adequate in evaluating animal carcinogenicity experiments.     

Weighted p-value adjustments for animal carcinogenicity trend test

A typical animal carcinogenicity experiment routinely analyzes approximately 10-30 tumor sites. Comparisons of tumor responses between dosed and control groups and dose-related trend tests are often evaluated for each individual tumor site/type separately. p-Value adjustment approaches have been proposed for controlling the overall Type I error rate or familywise error rate (FWE). However, these adjustments often result in reducing the power to detect a dose effect. This paper proposes using weighted adjustments by assuming that each tumor can be classified as either class A or class B based on prior considerations. The tumors in class A, which are considered as more critical endpoints, are given less adjustment. Two weighted methods of adjustments are presented, the weighted p adjustment and weighted alpha adjustment. A Monte Carlo simulation shows that both weighted adjustments control the FWE well. Furthermore, the power increases if a treatment-dependent tumor is analyzed as in class A tumors and the power decreases if it is analyzed as in class B tumors. A data set from a National Toxicology Program (NTP) 2-year animal carcinogenicity experiment with 13 tumor types/sites observed in male mice was analyzed using the proposed methods. The modified poly-3 test was used to test for increased carcinogenicity since it has been adopted by the NTP as a standard test for a dose-related trend. The unweighted adjustment analysis concluded that there was no statistically significant dose-related trend. Using the Food and Drug Administration classification scheme for the weighted adjustment analyses, two rare tumors (with background rates of 1% or less) were analyzed as class A tumors and 11 common tumors (with background rates higher than 1%) as class B. Both weighted analyses showed a significant dose-related trend for one rare tumor.     

The carcinogenicity prediction and battery selection (CPBS) method: a Bayesian approach

Recently, a large number of relatively inexpensive in vitro short-term tests have been developed to help predict the carcinogenicity of chemicals. The carcinogenicity prediction and battery selection (CPBS) method utilizes the results of such short-term tests to screen for chemicals that are most likely to cause cancer. The method is an integrated approach for analyzing large, often sparsely filled, data bases containing short-term test results, which often have only marginal representation of known non-carcinogens. The CPBS method is developed for the purpose of (i) determining the reliability and predictive capability of individual and batteries of short-term tests, and (ii) developing a strategy for formulating and selecting optimally preferred batteries of short-term tests for screening chemicals for further testing. The term 'optimally preferred' connotes the best acceptable combination of tests in terms of trade-offs among the multiple attributes of each test and resulting battery (e.g., cost, sensitivity, specificity, etc). The CPBS method consists of 5 major tasks: (1) data consolidation, (2) parameter estimation, (3) predictivity calculation, (4) battery selection and (5) risk assessment. Although there is a great need for more research and improvement, the CPBS method at its present stage should add an important method to the maze of the thousands of new chemicals that are introduced into drugs, foods, consumer goods and to the environment every year. This method should also provide an enhanced identification procedure for classifying chemicals more accurately as suspected carcinogens or non-carcinogens.     

Standardized tumor rates for chronic bioassays

If crude experimental proportions of animals with tumors from chronic bioassays for carcinogenicity are used for low-dose extrapolation in a risk analysis, different dose-specific patterns of mortality due to competing risks can bias the results. In order to adjust tumor rates for differential mortality across dose groups, Farmer, Kodell, and Gaylor (1982, Risk Analysis 2, 27-34) recommended using nonparametric estimates of probability distributions of times to onset of tumors, with competing causes of death removed, when performing a risk analysis. This paper extends the approach of Farmer et al. by proposing a method for adjusting tumor rates to reflect lifetime or near-lifetime tumor incidences that would be obtained if all dose groups experienced the control mortality rate from causes other than the tumor of interest. Thus, natural mortality due to competing risks is explicitly included, rather than removed. The proposed standardized tumor rates are calculated as a summation of adjusted age-specific probabilities of dying with a tumor during the course of an animal bioassay for carcinogenicity plus the probability of being alive with a tumor at the terminal sacrifice.     

Analysis of multiple tumor data from a rodent carcinogenicity experiment

Rodent tumorigenicity experiments are conducted to determine the safety of substances for human exposure. The carcinogenicity of a substance is generally determined by statistical tests that compare the effects of treatment on the rate of tumor development at several body sites. The statistical analysis of such studies often includes hypothesis testing of the dose effect at each of the sites. However, the multiplicity of the significance tests may cause an excess overall false positive rate. In consideration of this problem, recent interest has focused on developing methods to test simultaneously for the treatment effect at multiple sites. In this paper, we propose a test that is based on the count of tumor-bearing sites. The test is appropriate regardless of tumor lethality or of treatment-related differences in the underlying mortality. Simulations are given which compare the performance of the proposed test to several other tests including a Bonferroni adjustment of site-specific tests, and the test is illustrated using the data from the large ED01 experiment.     

Banding carcinogenic risks in developed countries: A procedural basis for qualitative assessment

Readily achieved comparative assessment of carcinogenic risks consequent upon environmental exposures may increase understanding and contribute to cancer prevention. Procedures for hazard identification and quantitative risk assessment are established, but limited when addressing novel exposures to previously known carcinogens or any exposure to agents having only suspected carcinogenic activity. To complement other means of data evaluation, a procedure for qualitative assessment of carcinogenic risk is described. This involves categorizing the relevant carcinogen and circumstances under which exposure occurs. The categories for carcinogens are those used for hazard identification and involve whether the agent is (1) a recognized carcinogen for humans; (2) probably or (3) possibly carcinogenic for humans; (4) characterized by inadequate evidence of carcinogenicity; or (5) lacking carcinogenicity. Exposure is categorized by whether it is one which (1) establishes the agent as a recognized carcinogen; (2) is taken into account in establishing carcinogenicity status; (3) is distinct from those providing clearest evidence of carcinogenicity; (4) is not characterized in relation to carcinogenicity; or (5) involves an exposure in which absence of carcinogenic outcome is observed. These two categories of evidence allow the risk inherent in a situation to be banded as indicative of a proven, likely, inferred, unknown or unlikely carcinogenic outcome, and further characterized using sub-bands. The procedure has been applied to about fifty situations. For recognized carcinogens, including asbestos and polycyclic aromatic hydrocarbons, risks consequent upon occupational exposure, the impact of point source pollution, residence near contaminated sites and general environmental exposure are allocated across the proven band and a likely sub-band. For solvents, pesticides and other compounds having less clearly established carcinogenicity, impact on residents living near a production site, or near earlier related industrial activity is allocated to certain inferred sub-bands. Unknown carcinogenic outcome, which identifies exposure to an agent with inadequate evidence of carcinogenicity rather than being indicative of equivocal or negative data in any context, indicates both the impact of certain pollutants and user-exposure to some consumer products. Situations allocated to the unlikely risk band principally involve certain consumer products. Overall, such risk assessment may be of greatest worth in focusing community attention on proven causes of cancer and associated preventive measures.     

Evaluation of Animal Carcinogenicity Studies: Cochran‐Armitage Trend Test vs. Multiple Contrast Tests

Typical animal carcinogenicity studies involve the comparison of several dose groups to a negative control. The uncorrected asymptotic Cochran‐Armitage trend test with equally spaced dose scores is the most frequently used test in such set‐ups. However, this test based on a weighted linear regression on proportions. It is well known that the Cochran‐Armitage test lacks in power for other shapes than the assumed linear one. Therefore, dichotomous multiple contrast tests are introduced. These build the maximum over several single contrasts, where each of them is chosen appropriately to cover a specific dose‐response shape. An extensive power study has been conducted to compare multiple contrast tests with the approaches used so far. Crucial results will be presented in this paper. Moreover, exact tests and continuity corrected versions are introduced and compared to the traditional uncorrected approaches regarding size and power behaviour. A trend test for any shape of the dose‐response relationship for either crude tumour rates or mortality‐ adjusted rates based on the simple Poly‐3 transformation is proposed for evaluation of carcinogenicity studies.     

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.     

A review of the genotoxicity of marketed pharmaceuticals

Information in the 1999 Physician's Desk Reference as well as from the peer-reviewed published literature was used to evaluate the genotoxicity of marketed pharmaceuticals. This survey is a compendium of genotoxicity information and a means to gain perspective on the inherent genotoxicity of structurally diverse pharmaceuticals. Data from 467 marketed drugs were collected. Excluded from analysis were anti-cancer drugs and nucleosides, which are expected to be genotoxic, steroids, biologicals and peptide-based drugs. Of the 467 drugs, 115 had no published gene-tox data. This group was comprised largely of acutely administered drugs such as antibiotics, antifungals, antihistamines decongestants and anesthetics. The remaining 352 had at least one standard gene-tox assay result. Of these, 101 compounds (28.7%) had at least one positive assay result in the pre-ICH/OECD standard four-test battery (bacterial mutagenesis, in vitro cytogenetics, mouse lymphoma assay (MLA), in vivo cytogenetics). Per assay type, the percentage of positive compounds was: bacterial mutagenesis test, 27/323 (8.3%); in vitro cytogenetics 55/222 (24.8%); MLA 24/96 (25%); in vivo cytogenetics 29/252 (11.5%). Of the supplemental genetic toxicology test findings reported, the sister chromatid exchange (SCE) assay had the largest percentage of positives 17/39 (43.5%) and mammalian mutagenesis assays (excluding MLA) had the lowest percentage of positives 2/91 (2.2%). The predictive value of genetic toxicology findings for 2-year bioassay outcomes is difficult to assess since carcinogenicity can occur via non-genotoxic mechanisms. Nevertheless, the following survey findings were made: 201 drugs had both gene-tox data and rodent carcinogenicity data. Of these, 124 were negative and 77 were equivocal or positive for carcinogenicity in at least 1 gender/1 species. Of the 124 non-carcinogens, 100 had no positive gene-tox findings. Of the remaining 24, 19 were positive in in vitro cytogenetics assays. Among the 77 compounds that exhibited equivocal or positive effects in carcinogenesis studies, 26 were positive in gene-tox assays and 51 were negative. Of the 51 negatives, 47 had multiple negative gene-tox assay results suggesting that these are probably non-genotoxic carcinogens. Statistical analyses suggested that no combination of gene-tox assays provided a higher predictivity of rodent carcinogenesis than the bacterial mutagenicity test itself.     

Cluster analysis in predicting the carcinogenicity of chemicals using short-term assays

Cluster analysis can be a useful tool for exploratory data analysis to uncover natural groupings in data, and initiate new ideas and hypotheses about such groupings. When applied to short-term assay results, it provides and improves estimates for the sensitivity and specificity of assays, provides indications of association between assays and, in turn, which assays can be substituted for one another in a battery, and allows a data base containing test results on chemicals of unknown carcinogenicity to be linked to a data base for which animal carcinogenicity data are available. Cluster analysis was applied to the Gene-Tox data base (which contains short-term test results on chemicals of both known and unknown carcinogenicity). The results on chemicals of known carcinogenicity were different from those obtained when the entire data base was analyzed. This suggests that the associations (and possibly the sensitivities and specificities) which are based on chemicals of known carcinogenicity may not be representative of the true measures. Cluster analysis applied to the total data base should be useful in improving these estimates. Many of the associations between the assays which were found through the use of cluster analysis could be 'validated' based on previous knowledge of the mechanistic basis of the various tests, but some of the associations were unsuspected. These associations may be a reflection of a non-ideal data base. As additional data becomes available and new clustering techniques for handling non-ideal data bases are developed, results from such analyses could play an increasing role in strengthening prediction schemes which utilize short-term tests results to screen chemicals for carcinogenicity, such as the carcinogenicity and battery selection (CPBS) method (Chankong et al., 1985).     

The Weight of Evidence Does Not Support the Listing of Styrene as “Reasonably Anticipated to be a Human Carcinogen” in NTP's Twelfth Report on Carcinogens

Styrene was listed as “reasonably anticipated to be a human carcinogen” in the twelfth edition of the National Toxicology Program's Report on Carcinogens based on what we contend are erroneous findings of limited evidence of carcinogenicity in humans, sufficient evidence of carcinogenicity in experimental animals, and supporting mechanistic data. The epidemiology studies show no consistent increased incidence of, or mortality from, any type of cancer. In animal studies, increased incidence rates of mostly benign tumors have been observed only in certain strains of one species (mice) and at one tissue site (lung). The lack of concordance of tumor incidence and tumor type among animals (even within the same species) and humans indicates that there has been no particular cancer consistently observed among all available studies. The only plausible mechanism for styrene-induced carcinogenesis—a non-genotoxic mode of action that is specific to the mouse lung—is not relevant to humans. As a whole, the evidence does not support the characterization of styrene as “reasonably anticipated to be a human carcinogen,” and styrene should not be listed in the Report on Carcinogens.     

Equivocal test results and prognostic staging uncertainties in the evaluation of patients with cancer of the prostate

In the staging of cancer, equivocal test results may occur in subjectively evaluated imaging procedures whose interpretations raise the possibility of metastases but are too uncertain to rule in or rule out metastatic spread, and in tests whose repetitions in the same patient yield conflicting results about dissemination. We assessed the frequency and prognostic correlates of test results giving equivocal evidence of disseminated (Stage IV) disease in an inception cohort of 280 patients receiving initial treatment for prostatic cancer between 1973-76. Among tests used for clinical staging, lymphangiograms (equivocal in 28 percent of tested patients), bone scans (equivocal in 25 percent of tested patients), and bone radiographs (equivocal in 20 percent of tested patients) most frequently yielded interpretations that equivocally suggested metastatic spread. Eighty-three (45 percent) of the 185 patients without clear-cut dissemination (Stages I-III) had at least one equivocal test result that suggested dissemination and that remained unresolved at the time of selection of therapy. Five-year survival (30 percent) for the 20 patients with local extracapsular spread (Stage III) and multiple equivocal results suggesting dissemination was identical to that for patients with clear-cut dissemination. In contrast, other patients with equivocal dissemination in Stages I-III had survival rates similar to those patients in the same stage and lacking equivocal dissemination. Unresolved equivocal staging results frequently complicate management decisions for patients with prostatic cancer. Survival analyses aid these decisions by demonstrating that equivocal findings of dissemination are prognostically unimportant unless they are multiple and occur in the context of unequivocal extracapsular spread.     

Estimation and Testing of Tumor Incidence Rates in Experiments Lacking Cause-of-Death Data

Approximate nonparametric maximum likelihood estimation of the tumor incidence rate and comparison of tumor incidence rates between treatment groups are examined in the context of animal carcinogenicity experiments that have interval sacrifice data but lack cause-of-death information. The estimation procedure introduced by MALANI and VAN RYZIN (1988), which can result in a negative estimate of the tumor incidence rate, is modified by employing a numerical method to maximize the likelihood function iteratively, under the constraint that the tumor incidence rate is nonnegative. With the new procedure, estimates can be obtained even if sacrifices occur anywhere within an interval. The resulting estimates have reduced standard error and give more power to the test of two heterogeneous groups. Furthermore, a linear contrast of more than two groups can be tested using our procedure. The proposed estimation and testing methods are illustrated with an experimental data set.     

Effects of treatment-induced mortality and tumor-induced mortality on tests for carcinogenicity in small samples

Statistical tests of carcinogenicity are shown to have varying degrees of robustness to the effects of mortality. Mortality induced by two different mechanisms is studied--mortality due to the tumor of interest, and mortality due to treatment independent of the tumor. The two most commonly used tests, the life-table test and the Cochran-Armitage linear trend test, are seen to be highly sensitive to increases in treatment lethality using small-sample simulations. Increases in tumor lethality are seen to affect the performance of commonly used prevalence tests such as logistic regression. A simple survival-adjusted quantal response test appears to be the most robust of all the procedures considered.     

Methods of identifying carcinogenic factors in medication, food and cosmetics]

The removal of carconogenic factors would be a most efficient measure to prevent cancer. As far as known chemicals are concerned, every effort is made to avert them, or at least to reduce the exposure to such compounds, but is necessary to detect unknown chemicals, especially those, drugs and foodstuffs for example, to which large populations are exposed. Giving suspected chemicals to laboratory animals is a standard carcinogenicity test. Studies of the carcinogenicity of unknown chemicals in animals are time consuming, expensive and cumbersome. This is why other means of establishing carcinogenicity are sought for. Several rapid tests are available to-day to select suspected carcinogens. These methods aim primarily at determining with chemicals--at the cell or tissue level--certain changes that would appear essential to trigger the carcinogenic process, such as somatic mutations. Studies are used on the mutagenicity of chemicals for bacteria of the Salmonella type, for yeast and cultured mammalian cells, together with the induction of recessive lethal mutations in Drosophila and of the unscheduled repair synthesis of DNA and the transformation of mammalian cells in vitro. Although there is an unequivocal correlation between the activity of chemicals in such tests and their carcinogenicity, discrepancies are found. Thus, the in vivo tests on laboratory animals remain the most reliable method to determine carcinogenicity. Whereas direct extrapolation of experimental data to human pathology is impossible, the experimental evidence of the carcinogenicity of any chemical should allow us to draw constructive conclusions. We shall never be able to reject drugs which produce the expected results and cannot be replaced by other drugs. But we can must the drugs whose beneficial effects are not exceptional and which can be replaced by other chemicals. As for the chemicals used in food additives and cosmetics, and recognized as carcinogenic in animals, they should be totally given up. Any decision made should be based on animal studies.     

Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/NTP

A survey has been conducted of 222 chemicals evaluated for carcinogenicity in mice and rats by the United States NCI/NTP. The structure of each chemical has been assessed for potential electrophilic (DNA-reactive) sites, its mutagenicity to Salmonella recorded, and the level of its carcinogenicity to rodents tabulated. Correlations among these 3 parameters were then sought. A strong association exists among chemical structure (S/A), mutagenicity to Salmonella (Salm.) and the extent and sites of rodent tumorigenicity among the 222 compounds. Thus, a approximately 90% correlation exists between S/A and Salm. across the 115 carcinogens, the 24 equivocal carcinogens and the 83 non-carcinogens. This indicates the Salmonella assay to be a sensitive method of detecting intrinsic genotoxicity in a chemical. Concordance between S/A and Salm. have therefore been employed as an index of genotoxicity, and use of this index reveals two groups of carcinogens within the database, genotoxic and putatively non-genotoxic. These two broad groups are characterized by different overall carcinogenicity profiles. Thus, 16 tissues were subject to carcinogenesis only by genotoxins, chief among which were the stomach, Zymbal's glands, lung, subcutaneous tissue and circulatory system. Conclusions of carcinogenicity in these 16 tissues comprised 31% of the individual chemical/tissue reports of carcinogenicity. In contrast, both genotoxins and non-genotoxins were active in the remaining 13 tissues, chief among which was the mouse liver which accounted for 24% of all chemical/tissue reports of carcinogenicity. Further, the group of 70 carcinogens reported to be active in both species and/or in 2 or more tissues contained a higher proportion of Salmonella mutagens (70%) than observed for the group of 45 single-species/single-tissue carcinogens (39%). 30% of the 83 non-carcinogens were mutagenic to Salmonella. This confirms earlier observations that a significant proportion of in vitro genotoxins are non-carcinogenic, probably due to their non-absorption or preferential detoxification in vivo. Also, only 30% of the mouse liver-specific carcinogens were mutagenic to Salmonella. This is consistent with tumors being induced in this tissue (and to a lesser extent in other tissues of the mouse and rat) by mechanisms not dependent upon direct interaction of the test chemical with DNA. Detection of 103 of the 115 carcinogens could be achieved by use of only male rats and female mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Testing for a trend in tumor prevalence rates: I. Nonlethal tumors

In the analysis of animal carcinogenicity studies, the standard survival-adjusted test for a dose-related trend in the prevalence of nonlethal tumors is the Hoel-Walburg test, which stratifies on age at death by grouping survival times into intervals. An alternative analysis assesses trend on the basis of the likelihood score test under a logistic model for the prevalence function, which adjusts for survival by including age at death as a continuous regression variable. Extensive simulations demonstrate that the test based on modeling the prevalence log-odds as a linear function of age is more powerful than the Hoel-Walburg test, regardless of the intervals used by the latter to stratify the data. Without incorporating a continuity correction, the size of each test often exceeds the nominal level, especially when the mortality patterns differ across dose groups. Corrected versions of the tests operate at conservative levels, where the degree of conservatism varies with the distribution of the data. When the mortality patterns for the dose groups are similar, both tests have essentially the same power to detect a trend in tumor prevalence rates. However, when mortality varies with dose, the logistic regression test with a linear age term is more powerful than the Hoel-Walburg test, and this gain in power increases as the dose-specific mortality patterns become more disparate.     

Age-adjusted exact trend tests in the event of rare occurrences

Preclinical animal carcinogenicity studies are usually concerned with testing the statistical significance of a dose-response relationship. When the response consists of a rare event such as the development of a certain type of tumor, exact statistical methods are often employed. The exact randomization trend test based on the multivariate hypergeometric distribution is less powerful in the presence of treatment-related risks other than the specified response. Particularly, the loss of power becomes more pronounced when competing risks cause progressively higher mortality rates with increasing dose, which is usual in practice. An age-adjusted form of the randomization test is proposed to adjust for this effect. Permutational distribution for Peto's cause-of-death (COD) test is also explored and compared with its asymptotic counterpart by simulation. The use of COD information has been a controversial issue due to the subjectivity in the pathologists' determinations as well as for economic reasons. The proposed age-adjusted exact test does not require COD, and it is shown to compare favorably to the COD tests via an extensive Monte Carlo simulation. Applications of the methods to two real data sets are included.     

Mutation tests in Neurospora crassa. A report of the U.S. Environmental Protection Agency Gene-Tox Program

Many mutation tests have been developed in Neurospora crassa during the almost 40 years of its use in mutation research. These tests detect two major classes of mutation: gene mutation and meiotic nondisjunction. Within the first class, forward- and reverse-mutation tests have been used. The forward-mutation tests include those that detect mutations at many loci and at specific loci. Both kinds of forward-mutation tests have been done in homokaryons (n) and heterokaryons (n + n'). From the publications that were not rejected by our pre-established criteria, data were extracted for 166 chemicals that had been tested for mutagenicity. Only 6 of the 166 chemicals have been tested in one or more gene mutation test and the meiotic nondisjunction test; these 6 chemicals were positive in the first and negative in the second. Of the 102 chemicals tested in one or more gene mutation tests, 94 were positive and 8 were negative. Of the 70 chemicals tested in the meiotic nondisjunction test, 7 were positive and 63 were negative. Two tests, the ad-3 forward-mutation test and the meiotic nondisjunction test, have been used most frequently. These two tests are especially important for hazard evaluation, because each detects a class of mutations that is likely to be deleterious or lethal in the F1 - disomics by the meiotic nondisjunction test and multilocus deletions by the ad-3 forward-mutation test in heterokaryons. Generally, direct-acting chemicals are mutagenic in the gene mutation tests, but few chemicals that required metabolic activation have been tested. Only 31 of the 166 chemicals tested in N. crassa have been tested for carcinogenicity. Among these chemicals, there is a good association between mutagenicity in gene mutation tests and carcinogenicity but a poorer association between meiotic nondisjunction and carcinogenicity; however, only a small number of chemicals has been tested in the meiotic nondisjunction test. Further use and development of certain mutation tests in N. crassa are desirable.     

Animal carcinogenicity studies: 1. Poor human predictivity

The regulation of human exposure to potentially carcinogenic chemicals constitutes society's most important use of animal carcinogenicity data. Environmental contaminants of greatest concern within the USA are listed in the Environmental Protection Agency's (EPA's) Integrated Risk Information System (IRIS) chemicals database. However, of the 160 IRIS chemicals lacking even limited human exposure data but possessing animal data that had received a human carcinogenicity assessment by 1 January 2004, we found that in most cases (58.1%; 93/160), the EPA considered animal carcinogenicity data inadequate to support a classification of probable human carcinogen or non-carcinogen. For the 128 chemicals with human or animal data also assessed by the World Health Organisation's International Agency for Research on Cancer (IARC), human carcinogenicity classifications were compatible with EPA classifications only for those 17 having at least limited human data (p = 0.5896). For those 111 primarily reliant on animal data, the EPA was much more likely than the IARC to assign carcinogenicity classifications indicative of greater human risk (p < 0.0001). The IARC is a leading international authority on carcinogenicity assessments, and its significantly different human carcinogenicity classifications of identical chemicals indicate that: 1) in the absence of significant human data, the EPA is over-reliant on animal carcinogenicity data; 2) as a result, the EPA tends to over-predict carcinogenic risk; and 3) the true predictivity for human carcinogenicity of animal data is even poorer than is indicated by EPA figures alone. The EPA policy of erroneously assuming that tumours in animals are indicative of human carcinogenicity is implicated as a primary cause of these errors.