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between toxicity (as quantified by the MTD) and carcinogenicity (as quantified by the TD50) is consistent with cell toxicity and the resulting cell proliferation's mediating of the carcinogenicity observed in some animal bioassays. However, the committee recognizes that other reasons for the observed relationship are possible. The committee suggests that experiments in which cell proliferation and carcinogenic or precarcinogenic responses are measured directly and compared will allow more definitive evaluation of relationships among toxicity, cell proliferation, and carcinogenicity.
RELATIONSHIP BETWEEN TOXICITY AND CARCINOGENICITY OBSERVED AT MTD
The practice of assessing risk associated with human exposures to chemicals on the basis of data from studies conducted in laboratory animals rests on a number of assumptions. Among them are the assumptions that the agents will produce qualitatively similar effects in animals and humans and that the relative potency in animals approximates the relative potency in humans. In general, assumptions about the relationships between animal and human data have proved fairly reliable. For instance, the application of toxicity, pharmacokinetic, and metabolic data derived from animal studies to human medicine has contributed to reducing the human risk associated with therapeutic agents.
The practice of classifying chemical substances as either carcinogenic or noncarcinogenic on the basis of animal tests conducted at the MTD involves a further assumption—that carcinogenesis is a specific response to exposure to specific chemical structures (agent specificity), rather than a nonspecific response of animals to induction of chronic toxicity. That assumption is necessary because chronic administration at the MTD often produces adverse effects in the tested animal populations. In fact, if no adverse effects have been observed in a chronic bioassay, the bioassay could be classified as inadequate, on the grounds that the MTD was not achieved and that the test had insufficient sensitivity to detect the carcinogenicity of the material tested. However, some researchers have argued that the observation of increased frequencies of tumors in animals receiving the MTD might not always be a chemical-specific
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phenomenon, but might be a secondary response to the induction of chronic toxicity. That is, perhaps chronic toxicity itself or some other high dose phenomenon is capable of inducing cancer.
It has been suggested in particular that carcinogenic responses to exposures at high doses are in many cases either totally or partially caused indirectly by mitogenesis (Ames and Gold, 1990). The idea is that high doses (at or near the MTD) cause toxic responses, which can cause cell proliferation (mitogenesis). A dividing cell is at greater risk of mutating than a quiescent cell, so mitogenesis is indirectly mutagenic and consequently associated with an increased likelihood of carcinogenesis. That mechanism might be totally responsible for a carcinogenic response, as hypothesized for sodium saccharin (Cohen and Ellwein, 1990a), or might accentuate the carcinogenicity of genotoxic compounds, as hypothesized for 2-acetylaminofluorene (2-AAF) (Cohen and Ellwein, 1990b). In the former case, a threshold was hypothesized for saccharin on the basis of chemical evidence that silicate crystals responsible for cell proliferation in rats do not form at lower doses. In the latter case, a synergistic effect between genotoxicity and cell proliferation was hypothesized for 2-AAF at high doses in the bladders of female mice, but only a genotoxic effect at lower doses at which cell proliferation was not expected to occur. That observation suggests a dose-response relationship for bladder cancer that is nonlinear at high doses but linear at lower doses where cell proliferation is absent. 2-AAF does not induce cell proliferation in all target organ systems, however; the dose-response relationship for liver cancer in mice appears to be linear throughout the entire dose range.
The relationship between toxicity (including mitogenesis) and carcinogenesis has been studied recently. A direct relationship between toxicity and carcinogenesis has been suggested for a number of nongenotoxic chemicals, such as saccharin (noted above), the antioxidant butylated hydroxyanisole (BHA), di-(2-ethylhexyl) phthalate (DEHP), and polychlorinated biphenyls (PCBs). Chronic rodent bioassays of those chemicals have revealed tumor induction at doses that also are associated with toxicity and the presence of nonneoplastic proliferative lesions. For example, in two-generation studies in adults and weanling rats (Anderson et al., 1988; Williams, 1988), saccharin administered at 5% of the diet induces bladder tumors, cytotoxicity, and regenerative hyperplasia, increasing the labeling index (a measure of cell proliferation) of the
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urothelium by a factor of 2-10. BHA induces forestomach carcinomas in rats and hamsters when administered as 2% of the diet; severe hyperplasia and cytotoxicity, as evidenced by erosion and ulceration, are also seen (Ito et al., 1991). DEHP is a peroxisome proliferator that has been shown to induce liver tumors, foci of hepatocellular alteration (previously described as neoplastic nodules), and an initial burst of mitosis in rats and mice when given at 3,000-12,000 ppm in the diet (Kluwe et al., 1982; Mitchell et al., 1985). Some PCB mixtures induce focal necrosis, fatty degeneration, and hyperplastic nodules in the livers of rats and mice at concentrations that also induce hepatic adenomas and carcinomas (Kimbrough and Linder, 1974; Kimbrough et al., 1975).
In addition to nongenotoxic carcinogens, genotoxic carcinogens induce toxicity, and consequent cell proliferation at higher, toxic doses might play a role in increasing tumor rates to beyond what would be expected from genotoxicity alone. For example, 2-AAF administration is associated linearly with DNANTP adduct formation in the mouse bladder; however, the tumor rate in that organ is consistent with the effects of an increased rate of cell proliferation at high doses that acts in combination with 2-AAF's genotoxicity to produce tumors (Cohen and Ellwein, 1990). A similar interactive effect between cell proliferation and tumorigenesis has been observed for benzo[a]pyrene applied to mouse skin (Albert et al., 1991). Epidemiologic evidence also supports an association between some kinds of chronic toxicity and cancer incidence, such as hepatitis and liver cancer, schistosomiasis and bladder cancer, tuberculosis and lung cancer, asbestosis and mesothelioma, and tropical ulcers and skin cancer (Preston-Martin et al., 1991). Explanations other than cellular proliferation (such as inflammation) are also possible.
Thus, there is evidence from various sources to support an association between toxicity and carcinogenesis. Several people have recently attempted to analyze the assumption that the phenomena are causally related. Hoel et al. (1988) and Tennant et al. (1991) have evaluated the relationships between mutagenicity, carcinogenicity, and toxicity in laboratory rodents with the NTP data base of chronic and, in some cases, subchronic bioassays performed on a total of 130 chemicals. In those bioassays, 50 rats and mice of each sex received the MTD, MTD/2, or MTD/4 for 2 years. Matched control groups were also used. Use of the data base provided an opportunity to compare the toxic properties of chemicals that were not carcinogenic with those of chemicals that
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were, under similar experimental conditions. Results included subchronic toxicity, neoplastic and chronic toxic effects observed after a 2 year exposure, chemical structure, and mutagenicity in salmonellae. Toxicity was defined by the investigators as ''any deleterious change in the tissues of animals exposed to chemicals that was discerned by histopathology"; most, but not all, toxic lesions were found to be associated with a proliferative response. Qualitative structural descriptors of toxicity were used to evaluate the relationships between regenerative or hyperplastic responses and cancer or the absence of cancer; rates of induced mitogenesis or increased rates of cell proliferation were not measured quantitatively and would have remained undetected in the absence of any structural change.
Table 2-2 shows the chemicals from the Tennant et al. (1991) study whose subchronic and chronic administration induced toxicity at the same site; some of the chemicals were carcinogens and some were not, but none was carcinogenic at the site of obvious toxicity. In contrast, Table 2-3 lists the chemicals that were carcinogenic at sites where both subchronic toxicity and chronic toxicity were present; about 40% of these were mutagenic. For both the concordant and discordant chemicals, most of the toxic lesions observed were proliferative, although the presence of proliferative lesions clearly is not predictive of carcinogenesis. Results of the Tennant et al. (1991) analysis and the Hoel et al. (1988) analysis indicate that some sites of toxicity of both carcinogens and noncarcinogens were associated with neoplasia and many were not. Some chemicals induced tumors at sites where toxicity was not in evidence, and some induced toxicity in some organs without inducing carcinogenesis. However, the majority of both mutagenic and nonmutagenic carcinogens induced tumors that were associated with chronic toxicity, although many of the same chemicals caused chronic toxicity at other sites that was not associated with carcinogenesis. Tennant et al. (1991) conclude that, although their results do not dissociate toxicity from the neoplastic process, they "illustrate the high degree of complexity of neoplastic processes and imply that there may be multiple mechanisms of carcinogenesis associated with even potent mutagens. They also provide a clear demonstration that chronic- exposure of rodents to chemicals that exhibit toxic effects does not necessarily result in carcinogenic effects. Further, even when chronic-exposures resulted in overt tissue specific toxicity, neoplasia did not necessarily develop." A temporary
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TABLE 2-2 Chemicals That Induced Both Subchronic and Chronic Toxicity but Not Carcinogenicity at Same Sitesa
Rats
Mice
Chemical
Site
Lesion
Site
Lesion
Noncarcinogens
2,4-Dichlorophenol
None
--
Liver
Syncytial alteration
Dimethoxane
Forestomach
Hyperplasia
Forestomach
Hyperplasia
Hydrochlorothiazide
Kidney
Nephropathy, mineralization
None
--
à -Methyldopa sesquihydrate
None
--
Kidney
Nephropathy, karyomegaly
Carcinogens
p-Chloroaniline HCl
Bone marrow, liver
Hyperplasia, hemosiderin
Kidney
Hemosiderin
Nitrofurantion
Testes
Degeneration
Testes
Degeneration
Tribromoethane
Liver
Inflammation, vacuolization
Liver
Inflammation, vacuolization
Malonaldehyde, Na salt
Glandular stomach; bone marrow
Ulcer, inflammation; hyperplasia
Pancreas
Atrophy
Furosemide
None
--
Kidney
Nephropathy
aFrom Tennant et al. (1991).
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TABLE 2-3 Chemicals That Induced Toxicity and Carcinogenicity at Same Sitea
Chemical
Mutagen
Rats
Mice
Site
Lesion
Site
Lesion
Glycidol
+
Forestomach
Dysplasia, carcinoma
Forestomach
Hyperplasia, carcinoma
p-Chloroaniline HCl
+
Spleen;
adrenal
Fibrosis, metaplasia, sarcoma; hyperplasia, pheochromocytoma
Liver
Hemosiderosis,
hepatocellular tumors
N,N-Dimethylaniline
+
Spleen
Fibrosis, metaplasia, sarcoma
Forestomach
Hyperplasia, squamous cell tumors
Nitrofurantoin
+
Kidney
Nephropathy, tubular cell adenoma and carcinoma
Ovary
Atrophy, ovarian tumors
4-Vinyl-1-cyclohexene diepoxide
+
Skin
Hyperplasia, basal and squamous cell carcinoma
Skin, ovary
Hyperplasia, basal and
squamous cell tumors;
atrophy, ovarian tumor
N-Methylolacrylamide
-
None
--
Ovary
Atrophy, granulosa cell tumors
Benzofuran
-
Kidney
Nephropathy, tubular cell adenocarcinoma
Liver; forestomach; lung
Syncytial changes, liver tumors;
hyperplasia, carcinoma; hyperplasia,
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Chemical
Mutagen
Rats
Mice
Site
Lesion
Site
Lesion
Ochratoxin A
-
Kidney
Degeneration, hyperplasia, tubular cell tumors
Not determined
Hexachloroethane
-
Kidney
Nephropathy, hyperplasia, tubular cell adenocarcinoma
Not determined
--
d-Limonene
-
Kidney
Mineralization, nephropathy, hyperplasia, tubular cell adenocarcinoma
None
--
Hydroquinone
-
Kidney
Nephropathy, tubular cell adenoma
Liver
Syncitial cell alteration, liver tumors
Phenylbutazone
-
Kidney
Papillary necrosis, nephropathy, transitional-cell carcinoma
Liver
Degeneration, hypertrophy, necrosis, liver tumors
aFrom Tennant et al. (1991).
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toxic condition's effect on carcinogenesis might not be detected with data from chronic or even subchronic bioassays; for example, it is possible that a chemical very early in the course of a bioassay induces toxicity that enhances its carcinogenic response, but that, because of an adaptive cellular response, no chronic proliferative lesions other than tumors develop. Nonetheless, the observations that have been reported after study of the NTP database support the existence of mechanisms of carcinogenesis more complex than simple mutation or induced cell proliferation; these mechanisms are yet to be identified.
Several other reports support the conclusion of an equivocal relationship between toxicity-induced proliferation and carcinogenesis discussed above. Wada et al. (1990) showed that p-methoxyphenol administered after initiation of rat forestomach tumors with N-methyl-N'-nitro-N-nitrosoguanidine caused epithelial damage and hyperplasia in a dose-dependent manner in the forestomach epithelium, but was not associated with any increase in tumors. In an investigation of the role of renal tubular cell hyperplasia in tumor promotion with barbital sodium (BBNa) after initiation with streptozotocin (STZ) in rats, STZ was found to reduce BBNa-induced nephropathy and cell proliferation without reducing renal tumor incidence (Konishi et al., 1990). The authors of the study note, however, that initiated cells might have a very different ability from noninitiated cells to respond to the mitogenic influences of a tumor promoter and that the reduction in overall DNA synthesis that was seen might be unrelated to the increased proliferation of preneoplastic or neoplastic cells. Ward et al. (1990) reached a similar conclusion in a study of the relationship between renal or hepatocellular hyperplasia and tumor promotion with di-(2-ethylhexyl)phthalate in mice initiated with N-nitrosoethylurea.
The observation that toxicity and carcinogenicity are not always detected simultaneously make it problematic to account for increased rates of cell proliferation that are associated with carcinogenesis when one performs risk assessments of either genotoxic or nongenotoxic chemicals. The greater-than-quadratic nature of many dose-response curves for mutagens tested at and below their MTDs and the observation of toxicity and proliferative lesions in the target organs of most mutagenic carcinogens suggest that mechanisms in addition to mutation are operative and, in particular, that enhanced cell proliferation is likely to be occurring and affecting the response. In addition, most nonmutagens
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also induce toxicity and nonneoplastic proliferative lesions at doses that also are associated with neoplasia. Any information on the dose-response nature of these effects, especially cell proliferation, should be included in assessments of risk where possible, although, as Konishi et al. (1990) and Ward et al. (1990) emphasize, the target cells for proliferative activity associated with carcinogenesis might not be the total parenchymal tissue; identification of the affected target cells, such as stem cells, could be necessary. These problems are addressed in the second part of this report, Issues in Risk Assessment: Two-Stage Models of Carcinogenesis.
In summary, the committee evaluated the likelihood that observed correlations between cancer potencies and other measures of toxicity of chemicals tested at the MTD are tautologous and result from bioassay design or the statistical methods used for analysis or have a biologic basis. The committee performed its evaluation by determining the correlation between estimates of the TD50 and the HDT of clearly carcinogenic chemicals found in the CPDB. A strong correlation between those quantities was observed, with no chemicals classifiable as having either high toxicity and low potency or low toxicity and high potency. The committee concluded that the correlation is partly tautologous because it applies only to chemicals with cancer potencies high enough to be detected in an MTD bioassay. However, the relationship is not entirely tautologous, possibly because the phenomena of toxicity and carcinogenesis have several similarities. The dichotomy is reflected in the conflicting results of Tennant et al. (1991), who reported that an association between some measures of toxicity and positive carcinogenicity results in the same target organ in some, but not all, NTP bioassays. It is not yet possible to draw further conclusions about the relationship between toxicity and cancer potency.
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