Mouse-specific carcinogens: an assessment of hazard and significance for validation of short-term carcinogenicity bioassays in transgenic mice

1. The International Conference on the Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for human use (ICH) has agreed that bioassay data from only one species, the rat, supported by appropriate mutagenicity and pharmacokinetic data and also information from new (unvalidated) short term in vivo screening tests for potential carcinogenicity, could be used for the licensing of human medicines. This proposal has been supported by reviews of the utility of testing pharmaceuticals in the mouse which have concluded that the mouse bioassay contributes little to regulatory decisions. The current review was undertaken to identify 'genuine' mouse-specific carcinogens using the Gold Carcinogenicity Potency Database (CPD) for the initial identification of potential mouse-specific carcinogens from published literature. Hazard assessments were completed for these chemicals with particular attention focused on the 'genuine' mouse-specific carcinogens. The significance of such chemicals has been discussed together with consideration of on-going work on the validation of short-term carcinogenicity bioassays using transgenic mice. 2. Seventy-six potential mouse specific carcinogens were identified through the Gold Carcinogenicity Potency Database. Following more detailed consideration a total of ten chemicals were excluded from further consideration (three were multispecies carcinogens, five were considered to be non-carcinogenic in the mouse, and the data for two were uninterpretable). The review focused on the remaining 66 chemicals. There was equivocal evidence of carcinogenicity to the rat for 28 chemicals and inadequate data for a further 23 chemicals. Fifteen 'genuine' mouse-specific carcinogens were identified. These 15 chemicals comprise two genotoxic mouse-specific carcinogens (N-methylolacrylamide (924-42-5), 2,6-Dichloro-p-phenylenediamine (609-20-1); five non-genotoxic mouse-specific carcinogens 2-Aminobiphenyl.HCl (2185-92-4), Captan (133-06-2), Dieldrin (60-57-7), Diethylhexyladipate (103-23-1), and Probenicid (57-66-9); five mouse-specific carcinogens with equivocal evidence of mutagenicity were identified; (2,4-diaminophenol.2HCl (137-09-7), Dipyrone (68-89-3), Ozone (10028-15-6), Vinylidene chloride (75-35-4), and Zearalenone (17924-92-4)), and three mouse-specific carcinogens with inadequate mutagenicity data (Benzaldehyde (100-52-7), Piperonyl sulphoxide (120-62-7), Ripazepam (26308-28-1)). 3. It is suggested that the two genotoxic mouse carcinogens would have been considered as potential carcinogens in the absence of a mouse bioassay. Of the five non-genotoxic mouse-specific carcinogens; three induced tumours in mouse liver only and are considered as being of low potential hazard to human health. The remaining two chemicals would have been missed in the absence of a mouse bioassay (2-aminobiphenyl (2185-92-4) and captan (133-06-2)) and thus are good candidates for evaluation in the short term bioassays in transgenic mice currently being validated. 4. The hardest group of mouse-specific carcinogens to evaluate are those for which there is equivocal or inadequate mutagenicity data. The difficulty in evaluating these particular chemicals emphasises the need for adequate mutagenicity data in addition to adequate carcinogenicity data in order to assess potential hazards to human health. Hazard assessments and a consideration of the potential role for short-term bioassays in transgenic mice for the eight chemicals in this subgroup are presented. 5. A number of general conclusions have been derived from this review. Firstly, there are insufficient published genotoxicity data to allow a full assessment fo mutagenic potential for 57/76 of the potential mouse-specific carcinogens identified from the CPD. This is surprising given the clear value of such data in interpreting bioassay results and the much greater resources required for carcinogenicity bioassays. (ABSTRACT TRUNCATED)     

Alternatives to the 2-species bioassay for the identification of potential human carcinogens

It is proposed that the standard 2-species rodent cancer bioassay protocol, as perfected by the US National Toxicology Program (NTP), has already fulfilled its most useful role by providing an unequalled carcinogenicity database by which to re-assess the type of carcinogen worthy of definition. Continued use of this resource and time consuming protocol can no longer be justified, except in rare circumstances of high and protracted human exposure to a chemical of unknown carcinogenicity. In those rare instances an enlarged bioassay of three or four test species should perhaps be considered, there being nothing fundamental about the rat/mouse combination. In the large majority of cases, however, a practical estimation of the carcinogenic potential of a chemical can be formed in the absence of lifetime carcinogenicity bioassay data. This can be achieved by its sequential study, starting with an appreciation of its chemical structure and anticipated reactivity and mammalian metabolism. After the shortterm evaluation of a range of additional properties of the agent, including its genetic toxicity, rodent toxicity and tissue-specific toxicity, confident predictions of the genotoxic and/or non-genotoxic carcinogenic potential of the agent can be made. In most situations these predictions will be suitable for framing hazard reduction measures among exposed humans. In some situations it may be necessary to evaluate these predicted activities using limited bioassays, a range of which are considered. Extensions of these limited carcinogenicity bioassays to a standard 2-year/2-species bioassay can only be supported in cases where the non-carcinogenicity of the agent becomes the important thing to define. The US NTP have evaluated the carcinogenicity of approximately 400 chemicals over the past 20 years, at a cost of hundreds of millions of US dollars. The experience gained by that and related initiatives, worldwide, can now be harnessed to classify thousands of priority chemicals as being either probable carcinogens or probable noncarcinogens. That can now be achieved using a fraction of the earlier resources and in a fraction of the time that would be required for the conduct of 2-species bioassays. The comfort factor for one group of people of the order of the present system, coupled to the comfort factor for another group of the delay in carcinogenicity assessment enforced by the present council of perfection, are the two main factors delaying transfer to a streamlined system for assessing the carcinogenic potential of chemicals to humans. A third delaying factor in the need for new and focused test data. Coordinated acquisition of such data could rapidly remove the first two obstacles.     

Prediction of carcinogenicity from two versus four sex-species groups in the carcinogenic potency database

Prediction of a positive result in rodent carcinogenesis bioassays using two instead of four sex-species groups is examined for the subset of chemicals in the Carcinogenic Potency Database that have been tested in four sex-species groups and are positive in at least one (n = 212). Under the conditions of these bioassays, a very high proportion of rodent carcinogens that are identified as positive by tests in four groups is also identified by results from one sex of each species (86-92%). Additionally, chemicals that are classified as "two-species carcinogens" or "multiple-site carcinogens" on the basis of results from four sex-species groups are also identified as two-species or multiple-site carcinogens on the basis of two sex-species groups. Carcinogenic potency (TD50) values for the most potent target site are similar when based on results from two compared to four sex-species groups. Eighty-five percent of the potency values are within a factor of 2 of those obtained from tests in 4 sex-species groups, 94% are within a factor of 4, and 98% are within a factor of 10. This result is expected because carcinogenic potency values are constrained to a narrow range about the maximum dose tested in a bioassay, and the maximum doses administered to rats and mice are highly correlated and similar in dose level. Information that can be known in advance of a 2-yr bioassay (mutagenicity, class, route, and maximum dose to test) does not identify groups of rodent carcinogens for which four sex-species groups are required to identify carcinogenicity. The range of accurate prediction of carcinogenicity using only male rats and female mice is 93% among mutagens and 88% among nonmutagens; for various routes of administration, 88-100%; for various chemical classes, 75-100%; and for various levels of the maximum dose tested, 81-100%. Results are similar for the pair male rats and male mice. Using a strength of evidence approach, weaker carcinogens are somewhat less likely than stronger carcinogens to be identified by two sex-species groups. Strength of evidence is measured using the proportion of experiments on a chemical that are positive, the extent to which tumors occur in animals that die before terminal sacrifice, and whether the chemical induces tumors at more than one site and in more than one species.     

The micronucleus test and NTP rodent carcinogens: not so many false negatives

In the study by Shelby et al. (1993) on 49 chemicals, the results of the micronucleus (MN) test in mouse bone marrow were compared with the results of the 2 year rodent carcinogenicity assays. Seven of the 25 rodent carcinogens were considered positive in the MN test, 5 following a protocol in which chemicals were given in three daily doses, and a further 2 when the chemical was administered only once. This low rate of positive results has led to disappointment in the MN test as a screen for carcinogens, but a careful examination of the data and of its analysis by Shelby et al. (1993) shows that many of the negative results are appropriate because: of the 18 carcinogens that were negative in the MN test, 1 has been retested and found to be non-carcinogenic, 9 were non-genotoxic and at least 2 were site-of-contact carcinogens not expected to be detected in the bone marrow. Two others were clearly positive in the MN test in other labs. Thus, the MN test 'missed' not 18 carcinogens, but 4 genotoxic carcinogens. The significance of these 4 needs further assessment, since three were liver specific carcinogens and the fourth was a very weak inducer of hemangiosarcomas in female mice only. Overall, the results of Shelby et al. (1993) do not cast such a shadow on the micronucleus test as many feared, and must be examined in the context of all the information available on each chemical. As Ashby and Tinwell emphasize in the accompanying article and in Tinwell and Ashby (1994), the data show that the MN test is capable of identifying human carcinogens and rodent germ cell mutagens, and remains a useful part of genotoxicity evaluation of chemicals.     

The L5178Y/tk+/- mouse lymphoma specific gene and chromosomal mutation assay a phase III report of the U.S. Environmental Protection Agency Gene-Tox Program

The L5178Y/tk+/- (-)3.7.2C mouse lymphoma assay (MLA) which detects mutations affecting the heterozygous thymidine kinase (tk) locus is capable of responding to chemicals acting as clastogens as well as point mutagens. Improvements in the assay to enhance detection of this spectrum of genetic events are summarized, and criteria for evaluating the data are defined. Using these criteria, the Phase III Work Group reviewed and evaluated literature containing MLA results published from 1976 through 1993. The data base included 602 chemicals of which 343 were evaluated as positive, 44 negative, 18 equivocal, 54 apparently inappropriate for evaluation in this test system with the published protocols, and 142 that were inadequately tested, and thus a definitive call could not be made. The overall performance of the assay is summarized by chemical class, and the outcome of testing 260 chemicals in the MLA is compared with Gene-Tox and National Toxicology Program evaluations of rodent carcinogenesis bioassay results for the same chemicals. Based on the Work Group's evaluation of published MLA data for chemicals that were considered adequately tested, it is concluded that for most chemicals the L5178Y/tk+/- mouse lymphoma assay is eminently well suited for genotoxicity testing and for predicting the potential for carcinogenicity.     

What do animal cancer tests tell us about human cancer risk?: Overview of analyses of the carcinogenic potency database

Many important issues in carcinogenesis can be addressed using our Carcinogenic Potency Database, which analyzes and standardizes the literature of chronic carcinogenicity tests in laboratory animals. This review is an update and overview of our analyses during the past 15 years, using the current database that includes results of 5152 experiments on 1298 chemicals. We address the following: 1. More than half the 1298 chemicals tested in long-term experiments have been evaluated as carcinogens. We describe this positivity rate for several subsets of the data (including naturally occurring and synthetic chemicals), and we hypothesize and important role in the interpretation of results for increased cell division due to administration of high doses. 2. Methodological issues in the interpretation of animal cancer tests: constraints on the estimation of carcinogenic potency and validity problems associated with using the limited data from bioassays to estimate human risk, reproducibility of results in carcinogenesis bioassays, comparison of lifetable and summary methods of analysis, and summarizing carcinogenic potency when multiple experiments on a chemical are positive. 3. Positivity is compared in bioassays for two closely related species, rats and mice, tested under similar experimental conditions. We assess what information such a comparison can provide about interspecies extrapolation. 4. Rodent carcinogens induce tumors in 35 different target organs. We describe the frequency of chemicals that induce tumors in rats or mice at each target site, and we compare target sites of mutagenic and nonmutagenic rodent carcinogens. 5. A broad perspective on evaluation of possible cancer hazards from rodent carcinogens is given, by ranking 74 human exposures (natural and synthetic) on the HERP indes.     

Regional activation of L-type voltage-sensitive calcium channels in experimental thiamine deficiency

At present (putative) human carcinogens are identified via epidemiological studies and testing using the chronic 2-yr rodent bioassay. Both methods have severe limitations in that they are slow, insensitive, expensive, and are also hampered by many uncertainties. The development of methods to modify specific genes in the mammalian genome has provided promising new tools for use in identifying carcinogens and characterizing their (qualitative) risk. Several transgenic mouse lines are currently under study to test their possible use in short-term carcinogenicity testing. One such candidate alternative transgenic model is the XPA knock-out mouse. These mice have an almost complete deficiency in DNA nucleotide excision repair (NER). Nevertheless, XPA-deficient mice are viable and have a background of a low incidence of spontaneous development of cancers. Approximately 15% of the mice develop hepatocellular adenomas (only after 1.5 yr). After treatment with ultraviolet-B radiation or 7,12-dimethylbenz(a)anthracene, the XPA-deficient mice developed squamous cell carcinomas and papillomas, respectively, on their skin. Oral treatment of XPA-deficient mice with benzo[a]pyrene (B[a]P), 2-acetylaminofluorene (2-AAF), and 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) resulted in lymphomas (B[a]P), liver and bladder tumors (2-AAF), and intestinal adenomas plus lymphomas (PhIP). These results look encouraging, but it should be noted that the compounds and agents tested thus far have all been substrate for nucleotide excision repair. Animal studies with different genotoxic or nongenotoxic compounds, as organized for instance within the framework of the International Life Sciences Institute/Health and Environmental Sciences Institute program, are needed to further evaluate the suitability of the XPA model for short-term carcinogenicity testing.     

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells

The standard method for assessing the carcinogenicity of lubricating oil base stocks is the mouse skin-painting bioassay. This assay has the advantage of directly measuring the endpoint of interest, dermal carcinogenicity, but has the drawback of being time-consuming and expensive. For this reason, a variety of biological and chemical assays have been developed as predictive alternatives to the in vivo assay. This publication describes the application of three such methods to the assessment of carcinogenic potential of hydrotreated, re-refined oils: the modified Ames test, the analytical determination of 3-7-ring polycyclic aromatic compound content and the 32P-postlabeling assay for DNA adduct induction.     

The origins of the neural crest. Part I: embryonic induction

Nearly 500 long-term rodent carcinogenicity studies carried out by the National Cancer Institute and the National Toxicology Program were examined, and 12 chemicals were identified that produced nasal tumors: allyl glycidol ether, p-cresidine, 1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 2,3-dibromo-1-propanol, dimethylvinyl chloride, 1,4-dioxane, 1,2-epoxybutane, iodinated glycerol, procarbazine, propylene oxide, and 2,6-xylidine. All 12 of these chemicals produced nasal tumors in rats, and 5 also produced nasal tumors in mice. Most of the nasal carcinogens (1) produced tumor increases in both sexes, (2) produced tumors at other sites as well, (3) had significantly reduced survival at doses that were carcinogenic, and (4) were genotoxic. Only 5 of the 12 nasal carcinogens were administered by inhalation. A variety of different types of nasal cavity tumors were produced, and specific tumor rates are given for those chemicals causing multiple tumor types. Increased incidences of nasal neoplasms were often accompanied by suppurative/acute inflammation, epithelial/focal hyperplasia and squamous metaplasia. However, high incidences of these nonneoplastic nasal lesions were also frequently seen in inhalation studies showing no evidence of nasal carcinogenicity, suggesting that in general nasal carcinogenesis is not associated with the magnitude of chronic toxicity observed at this site.     

Mechanical bridge to myocardial recovery

Assessment of the carcinogenic potential of chemical agents continues to rely primarily upon the chronic rodent bioassay, a resource-intensive exercise. Recent advances in transgenic technology offer a potential resource conserving approach to carcinogen detection. Incorporation of oncogenes with known roles in the development of neoplasms into the genomes of laboratory rodents may provide new models with the potential of quickly and accurately separating carcinogenic from noncarcinogenic chemicals. The insertion of the v-Ha-ras oncogene into the genome of FVB/N mice imparts the qualities of genetically initiated skin in the transgenic mouse line designated as Tg.AC. The skin of either hemizygous (animals carrying the transgene on 1 allele) or homozygous (transgene copies on both alleles) Tg.AC mice promptly responds to the application of nongenotoxic carcinogens, such as the classical tumor promoting phorbol esters, with the development of squamous papillomas. Tumor production generally begins after 8-10 applications of 2.5 micrograms/mouse (3 times/wk) of 12-O-tetradecanoylphorbol 13-acetate (TPA). Maximal tumor response is usually in evidence within 20 wk. If this transgenic mouse line is to be useful in the identification of carcinogenic chemicals, experimental protocols must be systematically optimized. Experiments were conducted to compare the relative responsiveness of male and female hemizygous and homozygous Tg.AC mice to the dermal application of TPA and the known human leukemogen, benzene. Results revealed shipment-related variabilities in the relative responsiveness of hemizygous male and female mice to the application of the proliferative agent. Homozygous mice of both sexes were more reliable and uniform in responsiveness to both TPA and benzene. Therefore, our standard protocol for the conduct of bioassays with the Tg.AC mouse line specifies the use of homozygous males and/or females.     

Janus carcinogens and mutagens

Janus carcinogens are carcinogenic agents that, under differing conditions of cell type or dose, can instead act as anticarcinogens. Studies by Haseman and Johnson [J.K. Haseman, F.M. Johnson, Analysis of rodent NTP bioassay data for anticarcinogenic effects, Mutat. Res. , 350 (1996) 131-142], have demonstrated that many chemicals that are carcinogenic for one tissue type can have anticarcinogenic action on another tissue type. As Magni et al. [G.E. Magni, R.C. von Borstel, S. Sora, Mutagenic action during meiosis and antimutagenic action during mitosis by 5-aminoacridine in yeast, Mutat. Res., 1 (1964) 227-230] have shown in 1964, this principle holds true for chemical mutagens as well, that is 9-aminoacridine is an antimutagen in the vegetative cell and a mutagen in the sporulating cell. The conclusion can be drawn that two established carcinogens, tobacco and ionizing radiation, are indeed Janus carcinogens. In their review of 'ambiguous carcinogens' (their name), Weinberg and Storer [A.M. Weinberg, J.B. Storer, Ambiguous carcinogens and their regulation, Risk Anal., 5 (1985) 151-156], pointed out that tobacco can be classified as an ambiguous carcinogen. The strong carcinogenicity and anticarcinogenicity of tobacco smoke and/or tobacco itself (i.e., chewing tobacco) may be due to components in the mixture, not that of a single carcinogenic chemical that also may be anticarcinogenic. Kondo [S. Kondo, Health Effects of Low-Level Radiation, Kinki Univ. Press, Osaka, Japan and Medical Physics Publishing, Madison, WI, 1995, 213 pp.] has compiled data that demonstrate that human populations who survive exposures to ionizing radiation generally live longer and have less cancer than unirradiated human populations, and this Janus phenomenon goes beyond the more trivial concept of increased sensitivity to radiation of rapidly dividing tumor cells. Thiabendazole is an interesting compound in that it is both aneugenic and antimutagenic, and yet it does not appear to be a carcinogen or a mutagen. It is discussed here because aneugenesis and antimutagenesis are at extremes of the mutagenic spectrum. In general, mutagenic or carcinogenic actions usually are at least partially understood at a molecular level, whereas antimutagenic and anticarcinogenic actions usually are not. It is possible there may be numerous specific mechanisms underlying the Janus activity of different chemicals.     

Newer oral contraceptives and the risk of venous thromboembolism

Over the past 5 years, a large collaborative study of chemically-induced mutation has been performed using the four bacterial strains Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101 in order to compare the specific spectrum of response to chemicals and to evaluate the usefulness (sensitivity) of each strain. Following the two collaborative studies to test the chemicals in category 1, chemicals previously judged as positive only in E. coli WP2 strains and derivatives of these chemicals, and category 2, oxidative agents or crosslinking agents, 22 compounds of category 3 consisting of 10 nonmutagenic carcinogens and another 12 chemicals were selected in this study. Twenty participating laboratories tested each compound in the same method as previous reports. In the group of nonmutagenic carcinogens, no chemical induced revertant colonies of any strain tested. In the group of other chemicals, response to the chemicals was similar in TA102 and WP2 uvrA/pKM101. Overall, in the three collaborative studies, a total of 79 compounds were tested. No difference in qualitative response to the four strains was observed for 71% (56/79) of the test chemicals. The combination of strains providing the greatest number of positive responses was WP2 uvrA/pKM101 with TA102; 84% (66/79) of the test chemicals elicited the same qualitative response in these two strains. Therefore, it is suggested that WP2 uvrA/pKM101 and TA102 can be included as a part of the standard tester strains for detection of mutagenic activity of chemicals.     

Synergistic enhancement of small and large intestinal carcinogenesis by combined treatment of rats with five heterocyclic amines in a medium-term multi-organ bioassay

The carcinogenic potential of five heterocyclic amines in combination was analyzed using a medium-term multi-organ bioassay. Male F344 rats were initially treated with five known carcinogens (diethylnitrosamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl)-nitrosamine, 1,2-dimethylhydrazine and 2,2'-dihydroxy-di-n-propylnitrosamine) over a 4 week period to induce preneoplastic changes in a variety of organs (wide spectrum initiation) and then given the five heterocyclic amines, all having the intestines as a target of their carcinogenicity, individually or in combination in the diet for a further 24 weeks. In the small and large intestines, simultaneous administration of five heterocyclic amines at doses 1/5 or 1/25 of those used in reported carcinogenicity studies resulted in higher incidences and multiplicities of adenocarcinomas than expected from the five individual effects, although the differences were not statistically significant. A synergistic effect based on the additive model was most evident (P < 0.141) with multiplicity data for carcinoma in the small intestine at the 1/25 dose. A similar trend was observed for Zymbal gland (P < 0.077), but not other carcinoma induction. Thus the results suggested that synergism depends on the carcinogenic organotropism of individual agents as well as the doses applied in combination.     

Strategies for evaluating carcinogenic substances

Cancer from exposure to chemicals is known for more than two centuries. Today, approximately 40 compounds have been identified as unequivocally carcinogenic in humans, more than 300 have been shown to be carcinogenic in animal experimentation. Accordingly, an old system subdivides carcinogens as human carcinogens (A1), animal carcinogens (A2, and compounds being suspective of exerting carcinogenic activity. There exist no threshoulds of effect for notorious carcinogens. In order to improve the protection of those exposed to carcinogens in the working area, a special type of tolerance values has been introduced (technical guidance values, TRK). Contrary to MAK-values, these TRKs take into account a certain residual cancer risk which in most cases can not be quantified. The amount of acceptable residual risks is a matter of political consensus which has to be organized between the societal groups involved. For the purpose of quantitative comparisons, "unit risks" have been introduced; the problematics of this category is discussed to some extend.     

Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group

The mouse has traditionally been used for the micronucleus test, with bone marrow the usual target organ. The aim of the 9th collaborative study by CSGMT was to evaluate the suitability of the rat for the micronucleus test, with bone marrow and peripheral blood as the target organ. Since the rat spleen eliminates circulating micronucleated erythrocytes, a rat peripheral blood micronucleus assay might not be feasible. Thirty-four Japanese laboratories and six overseas laboratories participated in this collaboration, and 40 chemicals were studied. As a rule, rat bone marrow and peripheral blood were analyzed using acridine orange staining. Among 36 mouse micronucleus-positive rat carcinogens, 34 of which had been evaluated by CSGMT, we observed 33 positive and three negative results with rat bone marrow and 30 positive, three equivocal, and three negative responses with rat peripheral blood. Of the two mouse micronucleus-negative rat carcinogens, acrylonitrile was positive in rat bone marrow and 4,4'-methylene bis(2-chloroaniline) was negative in both rat bone marrow and peripheral blood. Two chemicals reported to be mouse micronucleus-negative and rat-positive, azobenzene and Solvent Yellow 14, and one chemical reported to be mouse-positive and rat-negative, 1,2-dimethylhydrazine, gave positive responses in rat bone marrow and peripheral blood. The concordance between bone marrow and peripheral blood with rats was 92%. The concordance between rat and mouse erythrocytes was 88%. We concluded that the rat micronucleus assay, using either bone marrow or peripheral blood, can be used as an alternative to the mouse micronucleus assay.     

An empirical examination of factors influencing prediction of carcinogenic hazard across species

This study was stimulated by a recent U.S. Environmental Protection Agency (EPA, 1994) statement in draft environmental carcinogen risk assessment guidelines: "Several kinds of observations from animal studies can contribute to the judgment whether animal responses indicate a significant carcinogenic hazard to humans." We have investigated each of these kinds of observation using the cancer bioassay data system database. We obtained concordances from rat to mouse (and vice versa) for various subgroups of chemicals as follows: chemicals that induced tumors at multiple sites, chemicals that induce cancer in both sexes, chemicals that display reduced latency, and chemicals increasing the rates of rare tumors. The concordances are much higher for these chemical subgroups than the chemical groups that induce tumor at a single site, in only one sex, or without reduced latency, respectively. Thus, our findings support some of the EPA's suggested factors.     

High dose levels are not necessary in rodent studies to detect human carcinogens

Guidelines for the conduct of rodent carcinogenicity studies stipulate that when the test substance is administered via the diet, its concentration need not exceed 5% of the diet. Since it is now apparent that human carcinogens are amongst the most potent of rodent carcinogens, it should be possible to detect accurately potential human carcinogens by using only relatively low dose levels in rodent studies. Our analysis of the potency of human carcinogens in rodent studies leads to the conclusion that, even after applying a safety factor of 10, there is no purpose in using dose levels higher than 500 mg/kg body weight or 1% in the diet.     

Comparison of methods of identifying Helicobacter hepaticus in B6C3F1 mice used in a carcinogenesis bioassay

In a long-term rodent bioassay evaluating the carcinogenicity of triethanolamine, there was equivocal evidence of carcinogenic activity in male B6C3F1 mice, based on a marginal increase in the number of hepatocellular adenomas and hepatoblastomas. Interpretation was complicated by the presence of Helicobacter hepaticus in selected silver-stained liver sections which also had histological evidence of karyomegaly and oval cell hyperplasia. An increase in numbers of liver tumors, as evidence of carcinogenic activity, was also noted in female mice. However, H. hepaticus was not considered a complicating factor, because the livers of the female mice did not have histological features compatible with H. hepaticus infection. A retrospective analysis of 51 liver tissue samples from the original carcinogenicity study was conducted to determine the incidence of H. hepaticus infection and to evaluate different diagnostic approaches for assessing the presence of H. hepaticus in livers lacking characteristic lesions. In an initial evaluation of seven mice with liver tumors, argyrophilic bacteria resembling H. hepaticus were observed in liver sections, associated with characteristic liver lesions of hepatocytic karyomegaly and oval cell hyperplasia. Frozen liver tissue was available from four of these mice; all were confirmed to be infected with H. hepaticus by culture and PCR. In a larger subsequent analysis using frozen liver tissues from 44 mice without characteristic hepatic lesions, H. hepaticus-specific DNA was amplified from the livers of 21 of 44 of the mice (47%), compared to 14 of 44 of the mice (32%) having H. hepaticus cultured from their frozen liver tumors. The results of H. hepaticus culture and H. hepaticus-specific PCR concurred (i.e., both positive and negative results) in 84% of the cases. Microscopic detection of immunofluorescence-labeled or silver-stained bacteria in liver sections was relatively insensitive compared to either culture or PCR detection. This study confirms the widespread prevalence of H. hepaticus in mice, its potential to confound experimental results, and the need to include diagnostic testing for H. hepaticus in a murine health monitoring program.     

The micronucleus test as part of a short-term mutagenicity test program for the prediction of carcinogenicity evaluated by 143 agents tested

To evaluate the usefulness of the micronucleus test as a short-term assay for the detection of carcinogens, the correlation between micronucleus test data for 143 chemicals and corresponding cancer data, has been analyzed. For comparison, analogous data from Ames's test have also been collected for the same chemicals. In a comparison of the micronucleus test and Ames's test it was found that they had about the same specificity (around 80%) and predictive value (around 90%), while there was a significant difference in sensitivity in favor of Ames's test. The difference in sensitivity could be partly explained by differences in metabolizing capacities of these two test systems. It is concluded that a more elaborate test procedure for the micronucleus test would increase that sensitivity of this test. The principal value of the micronucleus test lies in the fact that it is an in vivo method, which may pick up effects at the chromosomal level not covered by bacterial assays. This is emphasized by the finding that the combination of Ames's test and the micronucleus test did increase the sensitivity of the screening procedure for the prediction of carcinogenic effects.     

The causes and prevention of cancer: the role of environment

The idea that synthetic chemicals such as DDT are major contributors to human cancer has been inspired, in part, by Rachel Carson's passionate book, Silent Spring. This chapter discusses evidence showing why this is not true. We also review research on the causes of cancer, and show why much cancer is preventable. Epidemiological evidence indicates several factors likely to have a major effect on reducing rates of cancer: reduction of smoking, increased consumption of fruits and vegetables, and control of infections. Other factors are avoidance of intense sun exposure, increases in physical activity, and reduction of alcohol consumption and possibly red meat. Already, risks of many forms of cancer can be reduced and the potential for further reductions is great. If lung cancer (which is primarily due to smoking) is excluded, cancer death rates are decreasing in the United States for all other cancers combined. Pollution appears to account for less than 1% of human cancer; yet public concern and resource allocation for chemical pollution are very high, in good part because of the use of animal cancer tests in cancer risk assessment. Animal cancer tests, which are done at the maximum tolerated dose (MTD), are being misinterpreted to mean that low doses of synthetic chemicals and industrial pollutants are relevant to human cancer. About half of the chemicals tested, whether synthetic or natural, are carcinogenic to rodents at these high doses. A plausible explanation for the high frequency of positive results is that testing at the MTD frequently can cause chronic cell killing and consequent cell replacement, a risk factor for cancer that can be limited to high doses. Ignoring this greatly exaggerates risks. Scientists must determine mechanisms of carcinogenesis for each substance and revise acceptable dose levels as understanding advances. The vast bulk of chemicals ingested by humans is natural. For example, 99.99% of the pesticides we eat are naturally present in plants to ward off insects and other predators. Half of these natural pesticides tested at the MTD are rodent carcinogens. Reducing exposure to the 0.01% that are synthetic will not reduce cancer rates. On the contrary, although fruits and vegetables contain a wide variety of naturally-occurring chemicals that are rodent carcinogens, inadequate consumption of fruits and vegetables doubles the human cancer risk for most types of cancer. Making them more expensive by reducing synthetic pesticide use will increase cancer. Humans also ingest large numbers of natural chemicals from cooking food. Over a thousand chemicals have been reported in roasted coffee: more than half of those tested (19/28) are rodent carcinogens. There are more rodent carcinogens in a single cup of coffee than potentially carcinogenic pesticide residues in the average American diet in a year, and there are still a thousand chemicals left to test in roasted coffee. This does not mean that coffee is dangerous but rather that animal cancer tests and worst-case risk assessment, build in enormous safety factors and should not be considered true risks. The reason humans can eat the tremendous variety of natural chemical "rodent carcinogens" is that humans, like other animals, are extremely well protected by many general defense enzymes, most of which are inducible (i.e., whenever a defense enzyme is in use, more of it is made). Since the defense enzymes are equally effective against natural and synthetic chemicals one does not expect, nor does one find, a general difference between synthetic and natural chemicals in ability to cause cancer in high-dose rodent tests. The idea that there is an epidemic of human cancer caused by synthetic industrial chemicals is false. In addition, there is a steady rise in life expectancy in the developed countries. Linear extrapolation from the maximum tolerated dose in rodents to low level exposure in humans has led to grossly exaggerated mortality forecasts. Such extrapo