National Institute of Environmental Health Sciences P. O. Box 12233 Research Triangle Park, NC 27709 E-mail: huff1{at}niehs.nih.gov.
To the Editor:
Two papers (Yoshihara et al., 2001; Devine et al., 2001
) in the August issue of Toxicological Sciences dealt with chemicals that have been tested for carcinogenicity by the National Toxicology Program (NTP). Both papers explored mechanisms of toxicity of these chemicals; the former dealt with metabolic activation of bisphenol A, and the latter explored mechanisms of ovarian toxicity (Hoyer et al., 2001
), both perhaps relevant to the carcinogenicity of these chemicals. Because neither of the articles mentioned these NTP bioassays, I wish to bring this additional and relevant information to the attention of your readers, especially with the renewed interest in bisphenol A, a well-known environmental endocrine disruptor chemical. The chemicals are bisphenol A and 4-vinylcyclohexene diepoxide, and for completeness the structurally related 4-vinylcyclohexene is also presented.
Bisphenol A
Bisphenol A (BPA), used in the manufacture of epoxy, polycarbonate, and polyester-styrene resins, is an environmental endocrine disruptor chemical. BPA was tested in long-term carcinogenesis studies in Fischer rats and B6C3F1 mice and the results reported in 1982 (NTP, 1982; Huff, 2001
). BPA was given in the diet for two years at 0, 1000, and 2000 ppm to groups of 50 male and 50 female rats; at 0, 1000, and 5000 ppm to male mice; and at 0, 5000, or 10000 ppm to female mice. In 1980 at a public peer review meeting, the NTP concluded, "Under the conditions of this bioassay, there was no convincing evidence that Bisphenol A was carcinogenic for F344 rats or B6C3F1 mice of either sex" (NTP, 1982
). However, the NTP also emphasized in the abstract, discussion, and conclusion that "The marginally significant increase in leukemia in male rats, along with an increase (not statistically significant) in leukemias in female rats and a marginally significant increase in the combined incidence of lymphomas and leukemias in male mice, suggests that exposure to bisphenol A may be associated with increased cancers of the hematopoietic system." Using the current NTP levels of evidence categories (Huff, 1992
) these findings would clearly be considered as "equivocal evidence" or possibly "some evidence" of carcinogenicity.
The NTP further opined in their abstract and conclusion: "A statistically significant increase in interstitial-cell tumors of the testes in male rats was also suggestive of carcinogenesis" and continued with this caveat "but was not considered to be convincing evidence of a compound-related effect because this lesion normally occurs at a high incidence in aging F344 rats." However, like the hematopoietic effects, this substantial and significant increase in testicular tumors in both exposure groups of male rats should likewise be considered as evidence of carcinogenicity: 35/49 (71%) in controls versus 48/50 (98%) in low dose and 46/49 (94%) in top dose (Haseman and Elwell, 1996;Huff, 2001
).
Additional estrogenic disruptor effects from exposing rodents to bisphenol A in these bioassays include an increasing trend for tumors of the mammary glands in male rats (an unusual tumor for males), and decreased trends for tumors of the adrenal gland in male (medulla) and female (cortical) rats and for endometrial stromal polyps of the uterus in female rats.
Overall, it appears that BPA exposure via the diet for two years should be considered associated with tumors of the hematopoietic system in rats and mice, and of the testes and of the mammary glands in male rats. Marginal decreases in tumors of the adrenal gland and of the uterus in rats should also be considered chemically related.
4-Vinylcyclohexene Diepoxide
4-Vinylcyclohexene diepoxide (4-VCHD) is used as a chemical intermediate and as a reactive diluent for diepoxides and epoxy resins. Two-year studies were conducted by administering 4-VCHD in acetone by dermal application, 5 days per week for 105 weeks to groups of 60 Fischer rats of each sex at 0, 15, or 30 mg/animal. Groups of 60 male and 60 female mice were exposed to 0, 2.5, 5, or 10 mg/animal on the same schedule for 103 weeks. Results of these bioassays were reviewed in public session in 1989, and were published shortly thereafter (Chhabra et al., 1990; IARC, 1994a
; NTP, 1989
).
Under the conditions of the NTP 2-year dermal studies, 4-VCHD induced both squamous cell and basal cell neoplasms of the skin of most male and female F344/N rats. In these 4-VCHDexposed rats there were no distant or non-application site tumors. In mice, 4-VCHD induced squamous cell carcinomas of the skin in most males and females. Regarding internally induced neoplasia, both benign and malignant tumors of the ovaries were caused by dermal exposure of 4-VCHD in female mice. Also in female mice, marginal increases of alveolar/bronchiolar adenomas/carcinomas of the lung may have been related to 4-VCHD.
4-Vinylcyclohexene
The structurally related 4-vinylcyclohexene (4-VCH) is a dimer of 1,3-butadiene (Huff et al., 1985) present in off-gasses from tire curing, Two year bioassays were conducted by administering 4-VCH in corn oil by gavage 5 days per week at doses of 0, 200, or 400 mg/kg body weight to groups of 50 male and female F344/N rats and B6C3F1 mice of each sex for 103 weeks. These studies were reviewed publicly in 1985, and published in 1986 (Collins et al., 1987
; IARC, 1994b
; NTP, 1986
).
Under the conditions of these 2-year gavage studies, 4-VCH induced benign and malignant tumors of the ovary in female mice. Also in female mice, increases of benign tumors of the adrenal glands may have been related to the administration of 4-VCH. The studies in male mice and both sexes of rats were considered inadequate bioassays of carcinogenicity because of extensive and early mortality and a lack of any conclusive evidence of a carcinogenic effect.
Nonetheless, and despite poor survival related to 4-VCH, there were some 4-VCH tumor target sites in these studies that deserve mention. In male mice there were marginal increases in malignant lymphomas and alveolar/bronchiolar adenomas/carcinomas of the lung; in rats increases were recorded for tumors of the clitoral gland in females and tumors of the skin in males. Interestingly, inflammation, epithelial hyperplasia, and ulcers of the forestomach were observed in top dose mice, yet no tumors were observed.
In summary, for each of these chemicals there was exposure-related neoplasia. BPA in the diet is considered reasonably associated with neoplasia of the hematopoietic system, testes, and mammary glands. Interestingly and structurally importantly, 4-VCH by gavage and 4-VCHD via skin shared some similar tumor target sites: ovary, lung, and skin. Of the nearly 500 chemicals tested by the NTP, only nine have been associated with tumors of the ovary. All of these have been seen in mice, with none in rats (Maronpot, 1987; Huff et al., 1991
; Davis, 1996
). 4-VCH, 4-VCHD, and the 4-VCH-monomer (1,3-butadiene) all caused ovarian tumors in mice, each by a different route of exposure: by oral (gavage) administration (4-VCH), by dermal application (4-VCHD), and by inhalation exposure (1,3-butadiene). These route-of-exposure findings add to and support the scientific view that chemical carcinogenesis is qualitatively independent of exposure route (Perera et al., 1989
; Huff, 1999
)
More details on these chemicals are available from the NTP Technical Reports and other cited references.
REFERENCES
Chhabra, R. S., Huff, J., Haseman, J., Jokinen, M. P., and Hetjmancik, M. (1990). Dermal toxicity and carcinogenicity of 4-vinyl-1-cyclohexene diepoxide in Fischer rats and B6C3F1 mice. Fundam. Appl. Toxicol.14, 752763.[ISI][Medline]
Collins, J. J., Montali, R. J., and Manus, A. G. (1987). Toxicological evaluation of 4-vinylcyclohexene. II. Induction of ovarian tumors in female B6C3F1 mice by chronic oral administration of 4-vinylcyclohexene. J. Toxicol. Environ. Health 21, 507524.[ISI][Medline]
Davis, B. J., Maronpot, R. R. (1996). Chemically associated toxicity and carcinogenicity of the ovary. In Cellular and Molecular Mechanisms of Hormonal Carcinogenesis: Environmental Influences. (J. Huff, J. Boyd, and J. C. Barrett, Eds.), pp. 285308. Wiley-Liss, New York.
Devine, P. J., Sipes, I. G., and Hoyer, P. B. (2001). Effect of 4-Vinylcyclohexene Diepoxide Dosing in Rats on GSH Levels in Liver and Ovaries. Toxicol. Sci.62, 315320.
Haseman, J. K., and Elwell, M. R. (1996). Evaluation of false positive and false negative outcomes in NTP long-term rodent carcinogenicity studies. Risk Anal. 16, 813820.[ISI][Medline]
Hoyer, P. B., Devine, P. J., Hu, X., Thompson, K. E., and Sipes, I. G. (2001). Ovarian toxicity of 4-vinylcyclohexene diepoxide: a mechanistic model. Toxicol. Pathol.29, 9199.[ISI][Medline]
Huff, J. (1992). A historical perspective on the classification developed and used for chemical carcinogens by the National Toxicology Program during 19831992. Scand. J. Work Environ. Health.18(Suppl.),7482.[ISI][Medline]
Huff, J. (1999).Value, validity, and historical development of carcinogenesis studies for predicting and confirming carcinogenic risks to humans. In. Carcinogenicity Testing, Predicting, & Interpreting Chemical Effects (K. T. Kitchin, Ed.) pp. 2:21123. Marcel Dekker, NY.
Huff, J. (in press). Carcinogenicity of bisphenol-A in Fischer Rats and B6C3F1 mice. Odontology.
Huff, J. E., Melnick, R. L, Solleveld, H. A., Haseman, J. K., Powers, M., Miller, R. A. (1985). Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalation exposure. Science227, 548549.[ISI][Medline]
Huff, J., Cirvello, J., Haseman, J., and Bucher, J. (1991). Chemicals associated with site-specific neoplasia in 1394 long-term carcinogenesis experiments in laboratory rodents. Environ. Health Perspect.93, 247270.[ISI][Medline]
IARC (1994a). 4-Vinylcyclohexene diepoxide. International Agency for Research on Cancer. Eval. Carcinog. Risks Hum. 60, 361372.
IARC (1994b). 4-Vinylcyclohexene. International Agency for Research on Cancer. Eval. Carcinog. Risks Hum. 60, 347359.
Maronpot, R. R. (1987). Ovarian toxicity and carcinogenicity in eight recent National Toxicology Program studies. Environ. Health Perspect. 73, 125130.[ISI][Medline]
NTP (1982). Carcinogenesis Bioassay of Bisphenol A (CAS No. 80057) in F344 Rats and B6C3F1 Mice (Feed Study). Tech. Rept No. TR-215. National Toxicology Program, Research Triangle Park, NC.
NTP (1986). Toxicology and Carcinogenesis Studies of 4-Vinylcyclohexene (CAS No. 100403) in F344/N Rats and B6C3F1 Mice (Gavage Studies). Tech. Rept No. TR-303. National Toxicology Program, Research Triangle Park, NC.
NTP (1989). Toxicology and Carcinogenesis Studies of 4-Vinyl-1-cyclohexene Diepoxide (CAS No. 106876) in F344/N Rats and B6C3F1 Mice (Dermal Studies). Tech. Rept No. TR-362. National Toxicology Program, Research Triangle Park, NC.
Perera, F., Brennan, T., and Fouts, J. R. (1989). Comment on the significance of positive carcinogenicity studies using gavage as the route of exposure. Environ. Health Perspect. 79, 315321.[ISI][Medline]
Yoshihara, S., Makishima, M., Suzuki, N., and Ohta, S. (2001). Metabolic activation of bisphenol A by rat liver S9 fraction.Toxicol. Sci . 62, 221227.