Nonlinearity and thresholds in dose-response relationships for carcinogenicity due to sampling variation, logarithmic dose scaling, or small differences in individual susceptibility.

Nonlinear and threshold-like shapes of dose-response curves are often observed in tests for carcinogenicity. Here, we present three examples where an apparent threshold is spurious and can be misleading for low dose extrapolation and human cancer risk assessment. Case #1: For experiments that are not replicated, such as rodent bioassays for carcinogenicity, random variation can lead to misinterpretation of the result. This situation was simulated by 20 random binomial samplings of 50 animals per group, assuming a true linear dose response from 5% to 25% tumor incidence at arbitrary dose levels 0, 0.5, 1, 2, and 4. Linearity was suggested only by 8 of the 20 simulations. Four simulations did not reveal the carcinogenicity at all. Three exhibited thresholds, two showed a nonmonotonic behavior with a decrease at low dose, followed by a significant increase at high dose ("hormesis"). Case #2: Logarithmic representation of the dose axis transforms a straight line into a sublinear (up-bent) curve, which can be misinterpreted to indicate a threshold. This is most pronounced if the dose scale includes a wide low dose range. Linear regression of net tumor incidences and intersection with the dose axis results in an apparent threshold, even with an underlying true linear dose-incidence relationship. Case #3: Nonlinear shapes of dose-cancer incidence curves are rarely seen with epidemiological data in humans. The discrepancy to data in rodents may in part be explained by a wider span of individual susceptibilities for tumor induction in humans due to more diverse genetic background and modulation by co-carcinogenic lifestyle factors. Linear extrapolation of a human cancer risk could therefore be appropriate even if animal bioassays show nonlinearity.

A mechanistic approach to modelling the risk of liver tumours in mice exposed to fumonisin B1 in the diet.

Data from the National Toxicology Program's carcinogenesis study of fumonisin B1 in B6C3F1 mice, conducted at the National Center for Toxicological Research, were used to fit the Moolgavkar-Venzon-Knudson (MVK) two-stage, clonal-expansion model of carcinogenesis. In addition to tumour data from the conventional 2-year bioassay, the study included data on tissue weights, cell proliferation, cell death, and sphingolipid metabolism in primary target organs. The model was used to predict 2-year liver tumour rates in female and male mice based on differences among dose groups in the effect of fumonisin B1 on the growth of normal tissue and on the proliferation of preneoplastic cells as a compensatory response to sphinganine-induced cell death. Fumonisin B1 was assumed to be non-genotoxic, i.e. the model did not include any effect of fumonisin B1 on either of the two mutation rates of the MVK model. The model was able to reproduce reasonably well the observed tumour rates in both female and male mice, predicting substantially increased rates above background only at the highest doses of fumonisin B1 in females.

Correlation of blood cholinesterase levels with toxicity of sarin in rats.

The dose-mortality response curve for sarin when administered to pregnant rats is extremely steep. The pregnant animal either died during the treatment or survived with no observable fetal toxicity. Animals that died displayed many symptoms characteristic of anticholinesterase toxicity. The present study was conducted to determine whether the maternal deaths, clinical observations, and/or weight loss could be correlated with baseline blood cholinesterase levels in individual animals. Cholinesterase levels (plasma and erythrocyte) were obtained prior to, during, and following treatment of nonpregnant rats by gavage with 380 microg/kg/d sarin for 10 d. After the first dose, there was a drop in the plasma cholinesterase levels, which then remained low throughout the dosing period. There was a statistically significant correlation between body weight loss and plasma cholinesterase levels of the sarin dosed animals. The surviving animals also had lower plasma cholinesterase levels and lower body weights, both of which recovered on the cessation of dosing. The erythrocyte cholinesterase levels were not different between treated and nontreated rats. Neither plasma or erythrocyte baseline cholinesterase levels nor relative or absolute cholinesterase decline values could be used as predictors of mortality from sarin administration in rats.

Dose-response trend tests for tumorigenesis adjusted for differences in survival and body weight across doses.

A relationship between rodent body weight and tumor incidence for some tissue/organ sites has been demonstrated in many studies. It is not uncommon for a chemical tested for carcinogenicity to also affect body weight due to toxicity and/or food consumption. In such cases, comparisons of tumor incidence may be biased by body weight differences across dose groups. A simple procedure was investigated for reducing this bias. This procedure divides the animals into a few groups on the basis of body weight. Body weight at 12 months was used, before the appearance of a tumor was likely to affect body weight. Statistics for dose-response trend tests are calculated within body weight strata and pooled to obtain an overall dose-response trend test. This procedure is analogous to stratifying animals on the basis of age at the time of removal from a study to account for differences in ages of animals across dose groups that can affect comparisons of tumor incidence. In this paper, differences in survival times of animals were adjusted by the Poly-3 technique used by the National Toxicology Program. This technique does not require the assignment of cause of death. Several examples from rodent chronic bioassays were investigated, where the high dose group had reduced body weights and associated reductions in tumor incidence. When we analyzed the data by body weight strata, some positive dose-response trends for tumor incidence were demonstrated. In one case, the body weight adjusted analysis indicated that a negative dose-response trend in tumor incidence was a real effect in addition to a body weight reduction. These examples indicate that it is important to consider the effects of body weight changes as low as 10%, and perhaps less, as possibly being caused by chemicals in 2-year bioassays for carcinogenesis. The simple procedure of analyzing tumor incidence within body weight strata can reduce the bias introduced by body weight differences across dose groups.

The use of Haber's law in standard setting and risk assessment.

Haber's Law simply states that the incidence and/or severity of a toxic effect depends on the total exposure, i.e. exposure concentration (c) rate times the duration time (t) of exposure (c x t). This rule, within constraints, is often used in setting exposure guidelines for toxic substances. Establishing reference doses (acceptable daily intakes) for long-term exposures when only the results of short-term studies are available requires the use of an uncertainty (safety) factor. The value of this uncertainty factor often approximates a value comparable to Haber's Law for extrapolation from short-term to long-term exposure durations. As a default procedure, cancer risk estimates are generally based on the average lifetime daily dose which is derived from the total cumulative exposure, i.e. Haber's (c x t). This has been shown both theoretically and empirically to be valid within a factor of 20 for carcinogenesis. This provides some credence for the use of an additional safety factor of 10, in some instances, for exposures of children to carcinogens. Finally, a generalization of Haber's Law, exposure concentration raised to a power times exposure duration, is discussed.

Percentiles of the product of uncertainty factors for establishing probabilistic reference doses.

Exposure guidelines for potentially toxic substances are often based on a reference dose (RfD) that is determined by dividing a no-observed-adverse-effect-level (NOAEL), lowest-observed-adverse-effect-level (LOAEL), or benchmark dose (BD) corresponding to a low level of risk, by a product of uncertainty factors. The uncertainty factors for animal to human extrapolation, variable sensitivities among humans, extrapolation from measured subchronic effects to unknown results for chronic exposures, and extrapolation from a LOAEL to a NOAEL can be thought of as random variables that vary from chemical to chemical. Selected databases are examined that provide distributions across chemicals of inter- and intraspecies effects, ratios of LOAELs to NOAELs, and differences in acute and chronic effects, to illustrate the determination of percentiles for uncertainty factors. The distributions of uncertainty factors tend to be approximately lognormally distributed. The logarithm of the product of independent uncertainty factors is approximately distributed as the sum of normally distributed variables, making it possible to estimate percentiles for the product. Hence, the size of the products of uncertainty factors can be selected to provide adequate safety for a large percentage (e.g., approximately 95%) of RfDs. For the databases used to describe the distributions of uncertainty factors, using values of 10 appear to be reasonable and conservative. For the databases examined the following simple "Rule of 3s" is suggested that exceeds the estimated 95th percentile of the product of uncertainty factors: If only a single uncertainty factor is required use 33, for any two uncertainty factors use 3 x 33 approximately 100, for any three uncertainty factors use a combined factor of 3 x 100 = 300, and if all four uncertainty factors are needed use a total factor of 3 x 300 = 900. If near the 99th percentile is desired use another factor of 3. An additional factor may be needed for inadequate data or a modifying factor for other uncertainties (e.g., different routes of exposure) not covered above.

Cancer risk estimation for mixtures of coal tars and benzo(a)pyrene.

Two-year chronic bioassays were conducted by using B6C3F1 female mice fed several concentrations of two different mixtures of coal tars from manufactured gas waste sites or benzo(a)pyrene (BaP). The purpose of the study was to obtain estimates of cancer potency of coal tar mixtures, by using conventional regulatory methods, for use in manufactured gas waste site remediation. A secondary purpose was to investigate the validity of using the concentration of a single potent carcinogen, in this case benzo(a)pyrene, to estimate the relative risk for a coal tar mixture. The study has shown that BaP dominates the cancer risk when its concentration is greater than 6,300 ppm in the coal tar mixture. In this case the most sensitive tissue site is the forestomach. Using low-dose linear extrapolation, the lifetime cancer risk for humans is estimated to be: Risk < 1.03 x 10(-4) (ppm coal tar in total diet) + 240 x 10(-4) (ppm BaP in total diet), based on forestomach tumors. If the BaP concentration in the coal tar mixture is less than 6,300 ppm, the more likely case, then lung tumors provide the largest estimated upper limit of risk, Risk < 2.55 x 10(-4) (ppm coal tar in total diet), with no contribution of BaP to lung tumors. The upper limit of the cancer potency (slope factor) for lifetime oral exposure to benzo(a)pyrene is 1.2 x 10(-3) per microgram per kg body weight per day from this Good Laboratory Practice (GLP) study compared with the current value of 7.3 x 10(-3) per microgram per kg body weight per day listed in the U.S. EPA Integrated Risk Information System.

New issues in cancer risk assessment.

When a nonlinear dose-response at low doses can be justified, an acceptable daily intake for a carcinogen can be obtained by dividing a benchmark dose, associated with a low incidence of tumors in animals, by uncertainty factors to account for animal-to-human extrapolation, human variability, and risk reduction from a low observed adverse-effect level. This approach can utilize mechanistic information to justify smaller uncertainty factors than typical default values of 10. If a nonlinear dose-response cannot be justified, traditional linear extrapolation from the benchmark dose to zero sometimes gives similar results. This suggests a unified risk-assessment procedure based on uncertainty factors. The issue of cross-species extrapolation based on the risk relative to background risks, rather than excess risk, is examined. The relative risk approach reduces the estimates of cancer risk in humans based on common rodent tumors, such as the liver in some strains of mice.

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Dose-response trend tests for tumorigenesis, adjusted for body weight.

Several studies have demonstrated a relationship between rodent body weight and tumor incidence for some tissue/organ sites. It is not uncommon for a chemical tested for carcinogenicity to also affect body weight. In such cases, comparisons of tumor incidence may be biased by body-weight differences across dose groups. A simple procedure was investigated for reducing this bias. This procedure divides the animals into a few groups based on body weight. Body weight at 12 months was used, before the appearance of a tumor was likely to affect body weight. Statistics for dose-response trend tests are calculated within body weight strata and pooled to obtain an overall dose-response trend test. This procedure is analogous to that currently used, of stratifying animals, based on their age at the time of removal from a study. Age stratification is used to account for differences in animal age across dose groups, which can affect comparisons of tumor incidence. Several examples were investigated where the high-dose group had reduced body weights and associated reductions in tumor incidence. When the data were analyzed by body-weight strata, some positive dose-response trends for tumor incidence were demonstrated. In one case, the weight-adjusted analysis indicated that a negative dose-response trend in tumor incidence was a real effect, in addition to a body weight reduction. These examples indicate that it is important to consider the effects of body weight changes as low as 10%, and perhaps below, that were caused by chemicals in 2-year bioassays for carcinogenesis. The simple procedure of analyzing tumor incidence within body-weight strata can reduce the bias introduced by weight differences across dose groups.

A unified approach to risk assessment for cancer and noncancer endpoints based on benchmark doses and uncertainty/safety factors.

A fundamental goal of toxicology is to determine safe levels of human exposure to toxic substances. In the absence of information to establish dose-response relationships at low exposure levels generally experienced by humans, high-dose to low-dose linear extrapolation is generally used for estimating carcinogenic risks and the no-observed-adverse-effect-level divided by uncertainty (safety) factors is widely used for establishing human exposure guidelines for noncancer effects. The basis and impact of this dichotomy is examined and questioned. It is proposed that a unified approach be adopted for establishing human exposure guidelines for both cancer and noncancer endpoints. It is suggested that a lower confidence limit on the dose estimated to produce an excess incidence of adverse health effects in 10% of the individuals in a human study or 10% of the animals in laboratory experiments be used as a point-of-departure. This dose would be divided by appropriate uncertainty factors to establish human exposure guidelines. For severe irreversible adverse health effects we suggest a total default uncertainty factor (divisor) for animal data on the order of 10,000, which is comparable to current guidelines. For reversible biological effects a smaller default uncertainty factor on the order of 1000 may be employed. This is comparable to the divisor often used currently when the point-of-departure is the lowest-observed-adverse-effect-level. It is asserted that the toxicological information generally available does not warrant numerical estimates of risk at low levels of human exposure. Rather, we support a unified approach for all adverse health effects of dividing a benchmark dose by appropriate uncertainty factors to establish guidelines for human exposures to toxic substances.

An FDA review of sulfamethazine toxicity.

Recently, changes have been proposed in the criteria historically used in the evaluation of the applicability to humans of some of the results obtained from the rodent carcinogenicity bioassay data. These questions center on the suitability of the rodent model for agents that exert their toxic effects via specific enzyme interactions and endocrine mechanisms which appear to be inoperative within humans. Within the U.S. Food and Drug Administration (FDA), this issue has been brought to the forefront of concern with the recent application for a New Animal Drug Application for sulfamethazine (SMZ). A panel of FDA experts from the National Center for Toxicological Research (NCTR), the Center for Veterinary Medicine (CVM), and the Center for Food Safety and Applied Nutrition has reviewed the sum of the scientific evidence available on the toxicology of SMZ. They noted that, in previous feeding studies at NCTR, high doses of SMZ were associated with significant incidences of thyroid tumors in mice and rats. The panel also notes that the tumorigenic activity of SMZ in rodents was due to its goitrogenic activity, resulting in constant stimulation of the thyroid by TSH. Humans, on the other hand, were found to be insensitive to the SMZ-like inhibition of thyroid function. Further, apart from X-irradiation and radioactive iodine, there are no other physical or chemical agents known to cause thyroid tumors in humans. Thus, the expert panel concludes that the best scientific information available indicates that elevated levels of TSH and the consequent thyroid tumors would not be produced under approved use conditions of SMZ. This conclusion is in agreement with recommendations made by three other panels, viz. the World Health Organization, the U.S. Environmental Protection Agency, and CVM, which also evaluated the public health risk of SMZ.

Combining uncertainty factors in deriving human exposure levels of noncarcinogenic toxicants.

Acceptable levels of human exposure to noncarcinogenic toxicants in environmental and occupational settings generally are derived by reducing experimental no-observed-adverse-effect levels (NOAELs) or benchmark doses (BDs) by a product of uncertainty factors (Barnes and Dourson, Ref. 1). These factors are presumed to ensure safety by accounting for uncertainty in dose extrapolation, uncertainty in duration extrapolation, differential sensitivity between humans and animals, and differential sensitivity among humans. The common default value for each uncertainty factor is 10. This paper shows how estimates of means and standard deviations of the approximately log-normal distributions of individual uncertainty factors can be used to estimate percentiles of the distribution of the product of uncertainty factors. An appropriately selected upper percentile, for example, 95th or 99th, of the distribution of the product can be used as a combined uncertainty factor to replace the conventional product of default factors.

Tumors and DNA adducts in mice exposed to benzo[a]pyrene and coal tars: implications for risk assessment.

Current methods to estimate the quantitative cancer risk of complex mixtures of polycyclic aromatic hydrocarbons (PAH) such as coal tar assume that overall potency can be derived from knowledge of the concentration of a few carcinogenic components such as benzo[a]pyrene (B[a]P). Genotoxic damage, such as DNA adducts, is thought to be an essential aspect of PAH-induced tumorigenesis and could be a biomarker for exposure useful for estimating risk. However, the role of B[a]P and the relationship of adduct formation in tumorigenesis have not been tested rigorously in models appropriate for human health risk assessment. Therefore, we directly compared tumor induction and adduct formation by B[a]P and coal tars in several experimental protocols, including one broadly accepted and used by regulators. We found that B[a]P content did not account for tumor incidences after exposure to coal tars. DNA adducts were found in both tumors and tumor-free tissue and tumor outcomes were not predicted by either quantitation of total DNA adducts or by the DNA adduct formed by B[a]P. These data suggest that risk assessments based on B[a]P content may not predict accurately risk to human health posed by environmental PAH.

Common mechanism of toxicity: a case study of organophosphorus pesticides.

The Food Quality Protection Act of 1996 (FQPA) requires the EPA to consider "available information concerning the cumulative effects of such residues and other substances that have a common mechanism of toxicity ... in establishing, modifying, leaving in effect, or revoking a tolerance for a pesticide chemical residue." This directive raises a number of scientific questions to be answered before the FQPA can be implemented. Among these questions is: What constitutes a common mechanism of toxicity? The ILSI Risk Science Institute (RSI) convened a group of experts to examine this and other scientific questions using the organophosphorus (OP) pesticides as the case study. OP pesticides share some characteristics attributed to compounds that act by a common mechanism, but produce a variety of clinical signs of toxicity not identical for all OP pesticides. The Working Group generated a testable hypothesis, anticholinesterase OP pesticides act by a common mechanism of toxicity, and generated alternative hypotheses that, if true, would cause rejection of the initial hypothesis and provide criteria for subgrouping OP compounds. Some of the alternative hypotheses were rejected outright and the rest were not supported by adequate data. The Working Group concluded that OP pesticides act by a common mechanism of toxicity if they inhibit acetylcholinesterase by phosphorylation and elicit any spectrum of cholinergic effects. An approach similar to that developed for OP pesticides could be used to determine if other classes or groups of pesticides that share structural and toxicological characteristics act by a common mechanism of toxicity or by distinct mechanisms.

Upregulation of apoptosis with dietary restriction: implications for carcinogenesis and aging.

The maintenance of cell number homeostasis in normal tissues reflects a highly regulated balance between the rates of cell proliferation and cell death. Under pathologic conditions such as exposure to cytotoxic, genotoxic, or nongenotoxic agents, an imbalance in these rates may indicate subsequent risk of carcinogenesis. Apoptotic cell death, as opposed to necrotic cell death, provides a protective mechanism by selective elimination of senescent, preneoplastic, or superfluous cells that could negatively affect normal function and/or promote cell transformation. The relative efficiency or dysfunction of the cell death program could therefore have a direct impact on the risk of degenerative or neoplastic disease. Dietary restriction of rodents is a noninvasive intervention that has been reproducibly shown to retard tumor development and most physiologic indices of aging relative to ad libitum-fed animals. As such, it provides a powerful model in which to study common mechanistic processes associated with both aging and cancer. In a recent study we established that chronic dietary restriction (DR) induces an increase in spontaneous apoptotic rate and a decrease in cell proliferation rate in hepatocytes of 12-month-old B6C3F1 DR mice relative to ad libitum (AL)-fed mice. This diet-induced shift in cell death/proliferation rates was associated with a marked reduction in subsequent development of spontaneous hepatoma and a marked increase in disease-free life span in DR relative to AL-fed mice. These results suggest that total caloric intake may modulate the rates of cell death and proliferation in a direction consistent with a cancer-protective effect in DR mice and a cancer-promoting effect in AL mice. To determine whether the increase in spontaneous apoptotic rate was maintained over the life span of DR mice, apoptotic rates were quantified in 12-, 18-, 24- and 30-month-old DR and AL mice. The rate of apoptosis was elevated with age in both diet groups; however, the rate of apoptosis was significantly and consistently higher in DR mice regardless of age. In double-labeling experiments, an age-associated increase in the glutathione S-transferase-II expression in putative preneoplastic hepatocytes in AL mice was rapidly reduced by apoptosis upon initiation of DR. Thus, intervention that promote a low-level increase in apoptotic cell death may be expected to protect genotypic and phenotypic stability with age. If during tumor promotion an adaptive increase in apoptosis effectively balances the dysregulated increase proliferation, the risk of permanent genetic error and carcinogenesis would be minimized.

U.S. Food and Drug Administration perspective of the inclusion of effects of low-level exposures in safety and risk assessment.

A brief overview is provided of some of the general safety and risk assessment procedures used by the different centers of the U.S. Food and Drug Administration (U.S. FDA) to evaluate low-level exposures. The U.S. FDA protects public health by regulating a wide variety of consumer products including foods, human and animal drugs, biologics, and medical devices under the federal Food, Drug, and Cosmetic Act. The diverse legal and regulatory standards in the act allow for the consideration of benefits for some products (e.g., drugs) but preclude them from others (e.g., food additives). When not precluded by statutory mandates (e.g., Delaney prohibition), the U.S. FDA considers both physiologic adaptive responses and beneficial effects. For the basic safety assessment paradigm as presently used, for example in the premarket approval of food additives, the emphasis is on the identification of adverse effects and no observed adverse effect level(s) (NOAEL). Generally, the NOAEL is divided by safety factors to establish an acceptable exposure level. This safety assessment paradigm does not preclude the consideration of effects whether they are biologically adaptive or beneficial at lower dose levels. The flexibility to consider issues such as mechanisms of action and adaptive and beneficial responses depends on the product under consideration. For carcinogenic contaminants and radiation from medical devices, the U.S. FDA considers the potential cancer risk at low exposure levels. This generally involves downward extrapolation from the observed dose-response range. The consideration of adverse effects of other toxicologic end points (e.g., reproductive, immunologic, neurologic, developmental) associated with low exposure levels is also becoming more of a reality (e.g., endocrine disrupters). The evaluation of the biologic effects of low-level exposures to toxic substances must include whether the effect is adverse or a normal physiologic adaptive response and also determine the resiliency of a physiologic system. The public health mandate of the U.S. FDA includes an active research program at the National Center for Toxicological Research and the other U.S. FDA centers to support the regulatory mission of the U.S. FDA. This includes the development of knowledge bases, predictive strategies, and toxicologic studies to investigate effects at the lower end of the dose-response range. Because of the wide diversity of legal and regulatory standards for various products regulated by the U.S. FDA agency-wide safety and risk assessment procedures and policies generally do not exist.

A comparison of the tumors induced by coal tar and benzo[a]pyrene in a 2-year bioassay.

The tumorigenicity of two coal tar mixtures was compared to that of benzo[a]pyrene after 2 years of feeding. Mixture 1, a composite of coal tar from seven coal gasification plant waste sites, was fed to female B6C3F1 mice (48 mice per group) for 2 years at doses of 0.0, 0.01, 0.03, 0.1, 0.3, 0.6 and 1.0%. Mixture 2, which was composed of coal tar from two of the seven waste sites and another site having a high benzo[a]pyrene content, was fed at doses of 0.0, 0.03, 0.1 and 0.3%. Additional groups of mice were fed 0, 5, 25 and 100 ppm benzo[a]pyrene. The coal tar diets induced a dose-related increase in hepatocellular adenomas and carcinomas, alveolar/bronchiolar adenomas and carcinomas, forestomach squamous epithelial papillomas and carcinomas, small intestine adenocarcinomas, histiocytic sarcomas, hemangiosarcomas in multiple organs and sarcomas. Benzo[a]pyrene treatment resulted in an increased incidence of papillomas and/or carcinomas of the forestomach, esophagus and tongue. A comparison of the results indicated that the benzo[a]pyrene in the coal tar diets could be responsible for the forestomach tumors. In contrast, the lung and liver tumors appeared to be due to other genotoxic components contained within the coal tar mixture, while the small intestine tumors resulted from chemically-induced cell proliferation that occurred at high doses of coal tar.