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8—
HAAs and Carcinogenesis in Animals

It has been hypothesized that environmental exposure to hormonally active agents (HAAs) results in an elevated risk of hormone-related cancers in humans. Human and experimental animal data link these cancers to exposure to endogenous hormones and diethylstilbestrol (DES) (see Appendix) (Herbst et al. 1971; Sonnenschein et al. 1974; Wiklund and Gorski 1982; Key and Pike 1988; Greenman et al. 1990; Brinton and Hoover 1993; Mittendorf 1995; Nandi et al. 1995). Endogenous estrogens have been associated with the development of tumors in the breast and endometrium in humans (Key and Pike 1988; Brinton and Hoover 1993; Nandi et al. 1995), and in the mammary, pituitary, and thyroid glands in animals (Sonnenschein et al. 1974; Wiklund and Gorski 1982; Greenman et al. 1990; Nandi et al. 1995). Endogenous estrogens could act as "initiators" by inducing DNA mutations (Liehr et al. 1986), as "promoters" by inducing cell proliferation (Russo and Russo 1978), or by allowing the persistence of tissues that normally regress or differentiate during development (Takasugi 1976: Bern 1992a). For a detailed discussion of the molecular mechanisms of estrogen-induced carcinogenesis, see Yager and Liehr (1996). The introduction of HAAs into the environment in the past 50-60 yr has preceded and overlapped the increasing incidence rates of some kinds of cancer. Because there is a lag period between exposure to a carcinogen and the induction of clinically apparent neoplasias, it is reasonable to investigate the association between HAAs and cancer.

This chapter reviews and evaluates data from animal studies relating environmental HAAs to cancers of the female and male reproductive systems and endocrine organs. The committee limited its review to cancer sites that are known from ancillary data to have some hormonal dependence and where activity should be most evident. However, the committee recognizes that some of the compounds discussed have been shown to cause cancer in other organ systems,continue



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Page 210 8— HAAs and Carcinogenesis in Animals It has been hypothesized that environmental exposure to hormonally active agents (HAAs) results in an elevated risk of hormone-related cancers in humans. Human and experimental animal data link these cancers to exposure to endogenous hormones and diethylstilbestrol (DES) (see Appendix) (Herbst et al. 1971; Sonnenschein et al. 1974; Wiklund and Gorski 1982; Key and Pike 1988; Greenman et al. 1990; Brinton and Hoover 1993; Mittendorf 1995; Nandi et al. 1995). Endogenous estrogens have been associated with the development of tumors in the breast and endometrium in humans (Key and Pike 1988; Brinton and Hoover 1993; Nandi et al. 1995), and in the mammary, pituitary, and thyroid glands in animals (Sonnenschein et al. 1974; Wiklund and Gorski 1982; Greenman et al. 1990; Nandi et al. 1995). Endogenous estrogens could act as "initiators" by inducing DNA mutations (Liehr et al. 1986), as "promoters" by inducing cell proliferation (Russo and Russo 1978), or by allowing the persistence of tissues that normally regress or differentiate during development (Takasugi 1976: Bern 1992a). For a detailed discussion of the molecular mechanisms of estrogen-induced carcinogenesis, see Yager and Liehr (1996). The introduction of HAAs into the environment in the past 50-60 yr has preceded and overlapped the increasing incidence rates of some kinds of cancer. Because there is a lag period between exposure to a carcinogen and the induction of clinically apparent neoplasias, it is reasonable to investigate the association between HAAs and cancer. This chapter reviews and evaluates data from animal studies relating environmental HAAs to cancers of the female and male reproductive systems and endocrine organs. The committee limited its review to cancer sites that are known from ancillary data to have some hormonal dependence and where activity should be most evident. However, the committee recognizes that some of the compounds discussed have been shown to cause cancer in other organ systems,continue

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Page 211 and in a variety of species. Compounds discussed in this chapter, with the exception of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been shown to possess estrogenic activity in at least one in vitro or in vivo bioassay (see Chapter 2). Finally, with the exception of DDT, it must be emphasized that this chapter focuses on postnatal exposures because no data are available on the carcinogenic effects of perinatal exposure to environmental HAAs to the F1 or succeeding generations. Bioassays Bioassays of the following compounds were evaluated with regard to carcinogenic effects in selected reproductive organs (i.e., endometrium/uterus, ovaries, testicles, and prostate gland) and endocrine organs (e.g., mammary, pituitary, thyroid, and adrenal glands): aldrin and dieldrin, 4,4'isopropylidenediphenol (bisphenol A), butyl benzyl phthalate (BBP), chlordecone, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), 1,1- dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), dichlorodiphenyltrichloroethane (DDT), endosulfan, endrin, lindane, methoxychlor, polychlorinated biphenyls (PCBs), TCDD, and toxaphene. Table 8-1 presents the experimental details and the specific results of the bioassays. A summary of those findings is presented below, and is followed by a discussion of their strengths and limitations. Negative results should be interpreted to mean that the HAA did not cause tumor formation under the conditions tested. Different test conditions may yield different results in the incidence of tumors. Aldrin and Dieldrin Aldrin was tested in bioassays in B6C3F1 and C3HeB/Fe mice, Osborne-Mendel rats, and mongrel dogs (Davis and Fitzhugh 1962; Fitzhugh et al. 1964; NCI 1978e). Dieldrin was tested in C3HeB/Fe, CF1, or B6C3F1 mice (Davis and Fitzhugh 1962; Thorpe and Walker 1973; Walker et al. 1973; NCI 1978e) Fischer 344, Osborne-Mendel, and Carworth Farm "E" strain (CF "E") rats (Fitzhugh et al. 1964; Walker et al. 1969; NCI 1978 e,f); Syrian Golden hamsters (Cabral et al. 1979); and beagle and mongrel dogs (Fitzhugh et al. 1964; Walker et al. 1969). None of the bioassays involved perinatal exposure. Overall, there was no evidence that either aldrin or dieldrin induced tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands. Bisphenol A Bisphenol A was tested for carcinogenicity in Fischer 344 rats and B6C3F1 mice (NTP 1982b). These tests involved exposure to adult animals only. An increase in testicular tumors (interstitial-cell tumors) was observed in 96% of thecontinue

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Page 212 rats of the low-dose group (p = 0.001) and in 94% of the high-dose group (p = 0.003), compared with 71% in the control group. However, the committee noted that aging Fischer 344 rats have a high incidence (more than 90%) of this type of tumor. Incidence data from the treated groups and the historical controls were not significantly different. No increases were found in the incidence of tumors of the endometrium/ uterus, ovaries, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands. Butyl Benzyl Phthalate Bioassays of BBP were conducted using Fischer 344/N rats (NTP 1997) and B6C3F1 mice (NTP 1982c). None of the studies involved prenatal exposure to BBP. There was no evidence that BBP increased the incidence of tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands compared with controls. Chlordecone Chlordecone was tested in bioassays using Osborne-Mendel rats and B6C3F1 mice (NCI 1976). Tests were conducted only on adult animals. There was no evidence that chlordecone increased the incidence of tumors of the endometrium/ uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands compared with controls. However, there is some evidence that chlordecone might induce cancers in the liver, which led the International Agency for Research on Cancer (IARC) to classify it as possibly carcinogenic to humans (IARC 1987). DDD DDD was evaluated for carcinogenicity in Osborne-Mendel rats and B6C3F1 mice (NCI 1978c). The tests involved exposure to adult animals only. DDD increased the incidence of thyroid tumors in male rats. The incidence of follicular-cell adenoma, follicular-cell carcinoma, c-cell adenoma, c-cell carcinoma, and adenoma was 50% in the low-dose group, 32% in the high-dose group, and 10% in control groups. The difference between the DDD-treated groups and the control groups was significant in the case of male rats with follicular-cell carcinoma and follicular-cell adenoma. There was no significant increase in the incidence of thyroid tumors in female rats. However, the authors indicate that the Cochran-Armitage test revealed a significant positive association between dose and combined incidence of follicular-cell adenomas and carcinomas in females. It is not known whether DDD induced the thyroid tumors through a hormonally mediated mechanism, although there is evidence that natural estrogens and DEScontinue

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Page 213 TABLE 8-1 Environmental HAAs and Cancer Species (Reference) Dose Resultsa and Limitations ALDRIN AND DIELDRIN Rat Osborne-Mendel Aldrin. Males and females (50 per Aldrin. No statistically significant increased incidence of tumors in reproductive or (NCI 1978e) group) received 30 or 60 ppm ( 1.5 or other endocrine organs. There was a significant positive linear trend in the incidence   3.0 mg/kg/d) in diet for 74 (males) or of follicular-cell adenoma or follicular-cell carcinoma of the thyroid in the male and   80 (females) wk. female low-dose groups when compared with the pooled control group, but not when     compared with the matched control group. Both male and female high-dose groups     failed to confirm the significance seen in the low-dose groups. Additionally, cortical     adenomas of the adrenal gland were observed in females in significant proportions     (p = .001 ) in the low-dose group, but not in the high-dose group, when compared to     the pooled control group. These increased incidences were not consistently     significant when compared to matched rather than pooled control groups.     Males. Mammary fibrosarcoma: 2%, 0%, 0%; testicular: NTR; prostate: NTR:     pituitary chromophobe adenoma and chromophobe carcinoma: 35%, 33%, 33%;     thyroid follicular cell adenoma, follicular cell carcinoma, c-cell adenoma. and c-cell     carcinoma: 48%, 29%, 71%; adrenal cortical adenoma, cortical carcinoma,     pheochromocytoma: 6%, 5%, 20%.     Females. Uterine leiomyosarcoma and endometrial stomal polyp: 13%, 21%, 0%;     mammary papillary adenocarcinoma: 20%, 14%, 30%, ovarian oranulosa cell tumor:     2%, 9%, 0%; pituitary chromophobe adenoma: 35%, 23%, 44%; thyroid follicular     cell adenoma. follicular cell carcinoma, c-cell adenoma, and c-cell carcinoma: 41%,     37%, 22%: adrenal cortical adenoma: 18%, 2%, 0%. Osborne-Mendel Dieldrin. Males and females Dieldrin. No statistically significant increased incidence of tumors in reproductive (NCI 1978e) (50 per group) received 29 or 65 ppm or other endocrine organs. Females showed a significant (p = .007) difference   (1.45 or 3.25 kg/mg/d) in diet for 80 between combined incidence of adrenal cortical adenoma or carcinoma in the   (low-dose group) or 59 (high-dose low-dose group and that in the pooled control group. However, the incidence in the   group) wk. high-dose group was not significant, and the incidences were not significant when     matched controls were used for comparison. Males did not show a statistically     significant difference between treated and control groups.     (table continues)

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Page 214 TABLE 8-1 Continued Species (Reference) Dose Results" and Limitations     Males. Mammary fibroma: 2%, 0%, 10%; testicular interstitial-cell tumor and     mesothelioma NOS: 4%, 0%, 0%; prostate: NTR; pituitary chromophobe adenoma,     chromophobe carcinoma, acidophil adenoma: 34%, 32%, 30%; thyroid follicular cell     adenoma, follicular cell carcinoma, c-cell adenoma, and c-cell carcinoma: 26%, 28%,     0%: adrenal cortical adenoma, pheochromocytoma, sarcoma NOS, ganglioneuroma:     2%, 11%, 10%.     Females. Uterine leiomyosarcoma and endometrial stromal polyp: 11%, 3%, 10%;     mammary adenoma NOS, adenocarcinoma NOS, fibroadenoma: 29%, 12%, 10%;     ovarian granulosa cell tumor: 4%, 2%, 0%; pituitary chromophobe adenoma,     chromophobe carcinoma: 27%, 27%, 50%; thyroid follicular cell adenoma, follicular     cell carcinoma, c-cell adenoma, and c-cell carcinoma: 37%, 34%, 25%; adrenal     cortical adenoma and carcinoma and pheochromocytoma: 13%, 8%, 0%. Fischer 344 Dieldrin. Males and females (24 per No statistically significant increased incidence of tumors in reproductive or other (NCI 1978f) group) received 2, 10, or 50 ppm endocrine organs. This study is limited because too few animals were used and also   (0.1, 0.5, or 2.5 mg/kg/d) in diet for the thyroids were not routinely examined microscopically.   104-105 wk.       Males. Mammary: NTR; testicular interstitial cell tumor: 96%, 100%, 83%, 100%:     prostate: NTR; pituitary adenoma: 13%, 41%, 4%, 0%; thyroid small cell carcinoma     and adenoma NOS: 0%, 0%, 100, 7%; adrenal gland: NTR.     Females. Uterine adenocarcinoma NOS, leiomyoma, endometrial stromal polyp,     endometrial stromal sarcoma: 80%, 63%, 46%, 59%; mammary adenoma, adeno-     carcinoma, cystadenoma, fibroma, fibroadenoma: 12%, 4%, 0%, 16%; ovary: NTR;     pituitary adenoma NOS: 30%, 17%, 9%, 8%; thyroid: NTR; adrenal gland: NTR. Osborne-Mendel Aldrin and Dieldrin. Males and No significant increased incidence of tumors in reproductive or other endocrine (Fitzhugh et al. females (12 per group) received 0.5. organs. This study is flawed because too few animals were used, the number of 1964) 2, 10, 50, 100, or 150 ppm (0.025, animals examined microscopically was limited, and there were high levels of early   0.1, 0.5 2.5, 5, 7.5 mg/kg/d) in diet mortality with insufficient numbers of animals surviving until termination of the   for 2 yr. study. (table continued on next page)break

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Page 215 (table continued from previous page)break Species (Reference) Dose Resultsa and Limitations Carworth Farm "E" Dieldrin. Males and females (25 No significant increased incidence of tumors in reproductive or other endocrine strain (Walker per group) received 0.1, 1, or 10 ppm organs. This study is flawed because the number of animals examined et al. 1969) (0.005, 0.05, or 0.5 mg/kg/d) in diet microscopically was limited. for 2 yr.     Mouse B6C3F1 Aldrin. Males (50 per group) Aldrin. No statistically significant increased incidence of tumors in reproductive or (NCI 1978e) received 4 or 8 ppm (0.6 or 1.2 other endocrine organs.   kg/mg/d) in diet for 80 wk. Females     (50 per group) received 3 or 6 ppm Males. Mammary: NTR; testicular interstitial cell tumor: 0%, 0%. 0%, 10%;   (0.45 or 0.9 kg/mg/d) in diet for prostate: NTR; pituitary: NTR; thyroid follicular cell adenoma: 13%, 2%, 0%,0%;   80 wk. adrenal gland: NTR.   Dieldrin. Males and females (50 Females. Uterine endometrial stromal polyp: 2%, 0%, 0%; mammary   per group) received 2.5 or 5 ppm leiomysarcoma: 0%, 0%, 10%; ovarian leiomysarcoma: 0%, 0%, 10%; pituitary   (0.37 or 0.75 mg/kg/d) in diet for chromophobe adenoma: 2%, 0%, 0%; thyroid adenoma: 2%, 0%, 0%; adrenal   80 wk. leiomysarcoma: 0%, 0%, 10%.     Dieldrin. No statistically significant increased incidence of tumors in reproductive     or other endocrine organs.     Males. Mammary: NTR; testicular: NTR; prostate: NTR; pituitary: NTR; thyroid:     NTR; adrenal gland: NTR.     Females. Uterine endometrial stromal polyp: 0%, 2%, 0%, 0%; mammary: NTR;     ovary: NTR; pituitary chromophobe adenoma: 0%, 3%, 3%, 0%; thyroid follicular     cell adenoma: 0%, 0%, 0%, 10%; adrenal gland: NTR. C3HeB/Fe Aldrin and Dieldrin. Males and No significantly increased incidence of tumors in reproductive or other endocrine (Davis and females (approximately 36 per group) organs. Fitzhugh 1962) received 10 ppm (1.5 mg/kg/d) in     diet for 2 yr.   Carworth Farm Dieldrin. Males and females (250- No significantly increased incidence of tumors in reproductive or other endocrine No. 1 (Walker et 400 per group) received 0.1, 1, or organs. al. 1973) 10 ppm (0.015, 0.15, 1.5 mg/kg/d)     in diet for 2 yr.       (table continues)

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Page 216 TABLE 8-1 Continued     Species (Reference) Dose Resultsa and Limitations Carworth Farm Dieldrin. Males and females (30 No significantly increased incidence of tumors in reproductive or other endocrine No. 1 (Thorpe and per group) received 10 ppm organs. Walker 1973) 1.5 mg/kg/d) in diet for 2 yr.   Hamster Dieldrin. Males and females (36 No significantly increased incidence of tumors in reproductive or other endocrine Syrian Golden per group) received 20, 60, or 180 organs. (Cabral et al. 1979) ppm (0.8, 2.4, or 7.2 mg/kg/d) in diet     for life span.   Dog Aldrin and dieldrin. Animals (sex No significant increased incidence of tumors in reproductive or other endocrine Mongrel not specified. 26 total) received organs. This study is flawed because too few animals were used and the length of (Fitzhugh et al. 8-400 ppm (0.2-10 mg/kg/d) in diet administration was not adequate for a carcinogenicity bioassay. 1964) for up to 25 mo.   Beagle Dieldrin. Males and females No significant increased incidence of tumors in reproductive or other endocrine (Walker et al. 1969) (5 per group) received 0.1 or 1 ppm organs. This study is flawed because too few animals were used and the length of   (0.005 or 0.05 mg/kg/d) in diet for administration was not adequate for a carcinogenicity bioassay.   2 yr.   BISPHENOL, A     Rat Males and females (50 per group) No statistically significant increased incidence of tumors in reproductive or other Fischer 344 received 1,000 or 2,000 ppm (50 endocrine organs. However, the incidence of mammary fibroadenomas in males was (NTP 1982b) mg/kg/d or 100 mg/kg/d) in diet for increased in the high-dose group compared with the control group (8% in the high- 103 wk.   dose group: 0% in the control group). That is a statistically significant dose-response     trend based on the Cochran-Armitage test (p = .015).     Males. Mammary fibroadenoma: 0%, 8%, 0%; testicular interstitial-cell tumors:     96%. 94%, 71%; pituitary carcinoma and adenoma: 27%, 31%, 28%; thyroid C-cell     adenoma and C-cell carcinoma: 12%, 13%, 19%; adrenal-gland cortical adenoma,     cortical carcinoma, and pheochromocytoma: 14%, 15%, 31%. (table continued on next page)break

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Page 217 (table continued from previous page)break Species (Reference) Dose Resultsa and Limitations     Females. Mammary adenoma: 2%, 0%, 0%; ovarian granulosa-cell tumor and     fibrosarcoma: 2%, 0%, 4%; pituitary adenoma: 41%, 50%, 52%; thyroid C-cell     adenoma and C-cell carcinoma: 13%c, 11%, 8%; adrenal-gland neoplasm, cortical     adenoma, pheochromocytoma, ganglioneuroma: 18%, 16%, 30%. Mouse Males (50 per group) received 1,000 No statistically significantly increased incidence of tumors in reproductive or other B6C3F1 or 5,000 ppm (150 mg/kg/d or 750 endocrine organs. However, the incidence of pituitary chromophobe carcinomas in (NTP 1982b) mg/kg/d) and females (50 per group) males was increased in the high-dose group compared with the control group (7% in   received 5,000 or 10,000 ppm (750 the high-dose group; 0% in the control group). That is a statistically significant   mg/kg/d or 1,500 mg/kg/d) in diet dose-response trend based on the Cochran-Armitage test (p = .016).   for 103 wk.       Males. Reproductive organs: NTR; pituitary chromophobe carcinoma: 0%, 7%, 0%;     adrenal cortical adenoma and sarcoma: 2%, 6%, 2%.     Females. Endometrial stromal polyp and leiomyosarcoma: 2%, 4%, 0%; mammary     adenocarcinoma and adenoma-squamous metaplasia: 2%, 2%, 0%; ovarian papillary     adenoma and granulosa-cell tumor: 0%, 4%, 0%; pituitary chromophobe adenoma and     chromophobe carcinoma: 0%, 3%, 5%; thyroid follicular-cell adenoma: 0%, 0%, 3%. BUTYL BENZYL PHTHALATE Rat Males (60 per group) received 3,000. No statistically significant increased incidence of tumors in reproductive or other Fischer 344/N 6,000, or 12,000 ppm (120, 240, or endocrine organs. Females exposed to the highest dose had an incidence of (NTP 1997) 500 mg/kg/d) in diet for 2 yr. fibroadenomas of the mammary gland that was statistically significantly decreased   Females (60 per group) received compared to control animals (p = .001). This decreased incidence was attributed to   6,000, 12,000, or 24,000 ppm (300, lower mean body weights in the dosed group.   600, 1200 mg/kg/d) in diet for 2 yr.       Males. Mammary carcinoma and fibroadenomas: 12%, 47% , 0%, 4%; testicular     adenocarcinoma (metastatic), interstitial cell adenoma: 92%, 98%, 90%, 88%;     prostate adenocarcinoma (metastatic): 0%, 2%,0%, 0%; pituitary adenoma and     carcinoma: 24%, 24%, 20%, 20%; thyroid c-cell adenoma, follicular cell adenoma,     follicular cell carcinoma: 8%, 6%, 8%, 10%; adrenal pheochromocytoma: 20%, 22%,     20%, 22%.     (table continues)

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Page 218 TABLE 8-1 Continued     Species (Reference) Dose Resultsa and Limitations     Females. Uterine deciduoma, leiomyoma, leiomyosarcoma, polyp stromal, sarcoma     stromal: 20%, 14%, 16%, 16%; mammary adenoma, carcinoma, and fibroadenomas:     62%, 66%, 22%, 65%; ovarian arrhenoblastoma NOS and granulosa cell tumor: 0%,     2%, 0%, 4%: pituitary adenoma and carcinoma: 52%, 52%. 26%, 45%; thyroid c-cell     adenoma, c-cell carcinoma, follicular cell adenoma, follicular cell carcinoma: 16%,     6%, 6%, 10%; adrenal ademona, carcinoma, ganglioneuroma, and     pheochromocytoma: 16%, 4%, 2%, 4%. Mouse Males and females (50 per group) No statistically significant increased incidence of tumors in reproductive or other B6C3F1 received 6,000 or 12,000 ppm (900 endocrine organs. (NTP 1982c) or 1,800 mg/kg/d) in diet for up to     103 wk. Males. Mammary: NTR; testicles: NTR; prostate: NTR; pituitary: NTR: thyroid     follicular cell adenomas and follicular cell carcinoma: 2%, 0%, 4%.     Females. Uterine leiomyoma, leiomyosarcoma, endometrial stromal polyp, and     endometrial stromal sarcoma: 0%, 6%, 6%; mammary: NTR, ovarian:     cystadenocarcinoma NOS: 0%. 2%, 0%; pituitary adenoma NOS: 0%, 0%, 2%;     thyroid: NTR: adrenal cortical adenoma: 2%, 0%, 0%. CHLORDECONE       Rat Males (50 per group) received 8 or No statistically significant increased incidence of tumors in reproductive or other Osborne-Mendel 24 ppm (0.4 or 1.2 mg/k/d) in diet endocrine organs. (NCI 1976) for 80 wk. Females (50 per group)     received 18 or 26 ppm (0.9 or 1.3 Males. Mammary fibroadenoma. adenoma, fibroma, adenocarcinoma, fibrolipoma:   mg/kg/d) in diet for 80 wk. 2%. 5%, 20%; testicular: NTR; prostate: NTR: pituitary chromophobe adenoma and     adenocarcinoma: 24%, 147%, 40%7; thyroid follicular cell carcinoma, follicular cell     adenoma, c-cell adenoma. c-cell carcinoma: 18%, 0%, 0%; adrenal cortical adenoma:     2%, 0%,0%.     Females. Uterine endometrial/stromal polyp, malignant lymphoma. squamous-cell     carcinoma: 8% , 4%; 0%; mammary fibroadenoma, adenoma, adenocarcinoma,     fibrolipoma: 14%, 4%, 50%; ovarian arrhenoblastoma and granulosa-cell tumor: 2%. (table continued on next page)break

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Page 219 (table continued from previous page)break Species (Reference) Dose Resultsa and Limitations     2%, 0%; pituitary chromophobe adenoma: 27%, 9%, 30%; thyroid follicular cell     carcinoma, follicular cell adenoma. c-cell adenoma, c-cell carcinoma: 6%, 7%, 0%;     adrenal cortical adenoma: 0%, 0%, 4%. Mouse Males (50 per group) received 20 or No statistically significant increased incidence of tumors in reproductive or other B6C3F1 23 ppm (3 or 3.45 mg/kg/d) in diet endocrine organs. (NCI 1976) for 80 wk. Females (50 per group)     received 20 or 40 ppm (3 or 6 Males. Mammary: NTR; testicular: NTR; prostate: NTR; pituitary: NTR; thyroid:   mg/kg/d) in diet for 80 wk. NTR; adrenal gland: NTR.     Females. Uterine/endometrial: NTR; mammary: NTR; ovarian cystadenoma: 0%.     2%, 0%; pituitary: NTR: thyroid: NTR; adrenal gland: NTR. DDD     Rat Males (50 per group) fed 1,647 ppm Increased incidence of thyroid tumors in males; positive association between dose Osborne-Mendel or 3,294 ppm (82.3 mg/kg/d or 164.7 and combined incidence of thyroid tumors in females. The high incidence of tumors (NCI 1978c) mg/kg/d) in diet for 78 wk. Females in control animals (37% mammary tumors, 21% pituitary tumors, 21% thyroid tumors   (50 per group) fed 850 ppm or in females) raises concern about the interpretation of this study.   1,700 ppm (42.5 mg/kg/d or 85.0     mg/kg/d) in diet for 78 wk. Males. Mammary fibroadenoma: 0%, 2%, 0%; testicle: NTR; prostate: NTR;     pituitary chromophobe adenoma and glioma: 27%, 24%, 5%; thyroid follicular-cell     adenoma, follicular-cell carcinoma, C-cell adenoma, C-cell carcinoma, and adenoma:     50%, 32%, 10%; adrenal-gland pheochromocytoma: 0%, 5%, 0%.     Females. Endometrial-uterine squamous-cell carcinoma: 3%. 3%, 0%; mammary     fibroadenoma and adenocarcinoma: 29%, 22%, 37%; ovary: NTR; pituitary     chromophobe adenoma: 47%, 36%. 21%; thyroid follicular-cell adenoma, follicular-     cell carcinoma, C-cell adenoma, and C-cell carcinoma: 31%, 22%, 21%; adrenal-     gland cortical adenoma, cortical carcinoma, and pheochromocytoma: 4%, 6%, 0%. Mouse Males and females (50 per group) fed No statistically significant increased incidence of tumors in reproductive or other B6C3F1 411 ppm or 822 ppm (61.6 mg/kg/d endocrine organs. (NCI 1978c) or 123.3 mg/kg/d) in diet for 78 wk.       Males. Mammary: NTR; testicle: NTR; prostate: NTR; pituitary: NTR; thyroid:     NTR; adrenal gland: NTR.     (table continues)

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Page 220 TABLE 8-1 Continued     Species (Reference) Dose Resultsa and Limitations     Females. Endometrial stromal polyp: 3%, 0%, 0%; mammary: NTR; ovary: NTR;     pituitary: NTR; thyroid: NTR; adrenal gland: NTR. DDE     Rat Males (50 per group) fed 437 ppm or No statistically significant increased incidence of tumors in reproductive or other Osborne-Mendel 839 ppm (21.8 mg/kg/d or 42.0 endocrine organs. The high incidence of tumors in control animals (30% mammary (NCI 1978c) mg/kg/d) in diet for 78 wk. Females tumors, 50% pituitary tumors, 15% thyroid tumors in females; 30% thyroid tumors in   (50 per group) fed 242 ppm or 462 males) raises concern about the interpretation of this study.   ppm (12.1 mg/kg/d or 23.1 mg/kg/d)     in diet for 78 wk. Males. Mammary adenoma and fibroadenoma: 0%, 2%, 5%: testicular interstitial-     cell tumors: 0%, 6%, 0%; prostate sarcoma: 0%. 6%, 0%; pituitary carcinoma and     chromophobe adenoma: 22%. 5%, 0%; thyroid follicular-cell adenoma, follicular-cell     carcinoma, C-cell adenoma, and C-cell carcinoma: 30%, 23%, 30%; adrenal gland:     NTR.     Females. Endometrial-uterine sarcoma, leiomysarcoma, and stromal polyp: 9%. 8%,     0%; mammary adenoma. adenocarcinoma, and fibroadenoma: 24%. 16%, 30%;     ovarian cystadenoma: 3%, 0%, 0%: pituitary carcinoma and chromophobe adenoma:     30%, 52%, 50%; thyroid follicular-cell adenoma, follicular-cell carcinoma, C-cell     adenoma, and C-cell carcinoma: 35%, 29%, 15%; adrenal-gland cortical adenoma:     3%, 4%, 0% . Mouse Males and females (50 per group) fed No statistically significant increased incidence of tumors in reproductive or other B6C3F1 148 ppm or 261 ppm (22.2 mg/kg/d endocrine organs. (NCI 1978c) or 39.1 mg/kg/d) in diet for 78 wk.       Males. Mammary: NTR; testicular interstitial-cell tumors: 2%, 0%, 0%; prostate:     NTR: pituitary: NTR; thyroid: NTR; adrenal gland; NTR.     Females. Endometrial-uterine adenocarcinoma, endometrial stromal polyp, and     hemanglioma: 0%, 4%, 6%; mammary adenocarcinoma and fibroadenocarcinoma: 4%,     0%, 5%, ovary: NTR pituitary: NTR, thyroid follicular cell carcinoma: 0%.     3%, 0%; adrenal gland: NTR. (table continued on next page)break

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Page 232 TABLE 8-1 Continued     Species (Reference) Dose Resultsa and Limitations     Males. Mammary carcinoma: 2%, 0%, 0%; testicle: NTR; prostate sarcoma: 0%,     3%, 0%; pituitary carcinoma, adenoma. and chromophobe adenoma: 31%, 16%, 43%;     thyroid follicular-cell adenoma, follicular-cell carcinoma, C-cell adenoma, and C-cell     carcinoma: 21%, 26%, 14%; adrenal-gland adenoma, cortical adenoma, cortical     carcinoma, and pheochromocytoma: 12%. 11%, 55%.     Females. Endometrial-uterine carcinoma, papillary adenoma, and stromal polyp:     24%, 13%, 0%; mammary adenoma, adenocarcinoma, papillary adenocarcinoma,     fibroma, fibroadenoma, and teratoma (malignant): 28%, 32%, 10%; ovarian carinoma     and granulosa-cell tumors: 3%, 3%, 0%; pituitary adenoma, chromophobe adenoma,     and chromophobe carcinoma: 37%, 59%, 38%; thyroid follicular-cell adenoma and     C-cell carcinoma: 2%, 17%, 17%; adrenal-gland cortical adenoma and cortical     carcinoma: 7%, 14%, 0%. Mouse Males and females (50 per group) fed No statistically significant increased incidence of tumors in reproductive or other B6C3F1 99 ppm or 198 ppm (14.8 mg/kg/d or endocrine organs. (NCI 1979a) 29.7 mg/kg/d) in diet for 80 wk.       Males. Mammary: NTR: testicle: NTR; prostate: NTR; pituitary: NTR; thyroid:     NTR; adrenal gland: NTR.     Females. Endometrial-uterine leiomyoma, stromal polyp: 4%,0%, 0%: mammary     adenoma: 2%, 0%, 0%; ovary: NTR: pituitary: NTR: thyroid hyperplasia (follicular     cell): 0%, 3%, 0%; adrenal gland: NTR. (table continued on next page)break

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Page 233 (table continued from previous page)break a Result values are shown in order of low-dose, mid-dose (if applicable), high-dose, and control groups. b Reuber (1978b) reanalyzed the data and reported an increased incidence of pituitary tumors in low dose rats (27% for males; 27% for females) compared to controls (0% for males; 4% for females). c Reuber ( 1981 ) reanalyzed the data and concluded that the pooled incidence of all tumors of the female genital tract was increased (62%) compared to controls (30%). d Reuber (1979) reanalyzed the data and reported a significant increase in the incidence of ovarian tumors in the low-dose (45%) and high-dose (57%) groups compared to controls (0%); a significant increase in pituitary tumors in the low-dose (37% in males; 59% in females) and high-dose groups (40% in males; 64 in females) compared to pooled controls (16% in males; 21% in females); a significant increase in the incidence of thyroid tumors in males of the low-dose group (42%) and in females of the low-dose (24%) and high-dose (28%) groups compared with pooled controls (18% for males; 7% for females); a significant increase in the incidence of adrenal gland tumors (malignant and benign) in males and females of the low-dose (35% in males; 45% in females) and high-dose (60% in males; 57 in females) groups compared to pooled controls (16% for males; 7% for females). e Reuber (1980) reanalyzed the data and reported an increased incidence of mammary gland tumors in female rats in the low-dose (33%) and high-dose (30%) groups compared to the control group (15%); a significant increase in the incidence of ovarian carcinomas in the low-dose (11%) and high-dose (23%) groups compared to controls (0%); twice as many adenomas and carcinomas of the pituitary in female rats of the high-dose group (43%) compared to controls (20%); an increased incidence of adrenal gland tumors in females of the low-dose (30%) and high-dose (38%) groups compared to controls (15%); an increase in thyroid tumors in treated rats (no data were provided). f NTR, no tumors reported.

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Page 234 induce follicular-cell adenoma in mice (Greenman et al. 1990). However, male and female mice fed DDD did not develop thyroid tumors. There was no evidence that DDD induced tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, pituitary gland, or adrenal glands. DDE DDE was tested for carcinogenicity in Osborne-Mendel rats and B6C3F1 mice (NCI 1978c). The tests involved exposure to adult animals only. There was no evidence in either species that DDE caused an increase in the incidence in tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, or adrenal glands. An increased incidence of pituitary tumors was observed in rats of the low- and high-dose groups compared with control rats, but the effect was not dose related. It should be noted that 50% of the female controls had pituitary tumors, which is a higher incidence than normally observed in the historical controls. No pituitary tumors were observed in the study with mice. DDT DDT was tested in many bioassays in two strains of rat (Osborne-Mendel and Carworth) (Fitzhugh and Nelson 1947; Treon and Cleveland 1955; Radomski et al. 1965: Deichmann et al. 1967; NCI 1978c), four strains of mouse (B6C3F1, BALB/c, CF1, and A strain) (Tarján and Kemény 1969; Shabad et al. 1973; Terracini et al. 1973; Thorpe and Walker 1973; Turusov et al. 1973; Walker et al. 1973; NCI 1978c), hamsters (Agthe et al. 1970; Graillot et al. 1975; Cabral et al. 1982; Rossi et al. 1983), monkeys (Durham et al. 1963; Adamson and Sieber 1983), and dogs (Lehman 1965). Four of the studies with mice were multigeneration studies that involved exposure to DDT during fetal life, lactation, and after weaning for either 6 mo (Tarján and Kemény 1969) or for their life span (Shabad et al. 1973; Terracini et al. 1973; Turusov et al. 1973). Two studies (Cabral et al. 1982; Rossi et al. 1983) reported an increased incidence of adrenal gland tumors (adrenocortical adenomas) in male and female hamsters. The increase was significant for the females. In two other studies with hamsters, DDT did not induce adrenal gland tumors, but early deaths occurred in one study (Agthe et al. 1970), and the length of administration was less than lifetime in the other (Graillot et al. 1975). In the bioassay conducted by NCI (1978c), DDT administration did not result in an increase in the incidence of adrenal gland tumors in rats or mice, but the Cochran-Armitage tests found a positive association between DDT and the incidence of pheochromocytoma in female rats. No increased incidence of adrenal gland tumors was observed in other bioassays with adult mice, rats, monkeys, or dogs or in multigeneration bioassays with mice exposed perinatally.break

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Page 235 There was no evidence that DDT increased the incidence of tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, or pituitary gland in animals treated prenatally or in adulthood. However, there is some evidence that DDT might induce cancers in other organs, such as the liver, which led IARC to classify it as possibly carcinogenic to humans (IARC 1991). A minor component of DDT—o,p'-DDT—is estrogenic at a dose of more than 25 mg/kg of body weight per day. That compound promoted the growth of MT2 mammary adenocarcinoma cells injected into ovariectomized Wistar-Furth rats (Robison et al. 1985b), which demonstrates that DDT can support the growth of an estrogen-responsive tumor. Endosulfan Endosulfan was tested in bioassays using Osborne-Mendel rats and B6C3F1 mice (NCI 1978b). The tests involved exposure to adult animals only. There was no evidence that endosulfan induced tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands, but endosulfan was toxic to male rats and mice. Endrin Bioassays of endrin were conducted with Osborne-Mendel rats (Deichmann et al. 1970; Reuber 1978a; NCI 1979b) and B6C3F1 mice (NCI 1979b). All of these studies involved exposure to adult animals. One study (Reuber 1978a) reported an increased incidence of mammary gland and thyroid tumors in Osborne-Mendel rats, but two other bioassays in rats did not find an increase in those types of tumors (Deichmann et al. 1970; NCI 1979b). A bioassay with B6C3F1 mice (NCI 1979b) was also negative. Reuber's criteria for classifying tissues as tumorigenic were not consistent with those of other investigators (EPA 1979b). There are data showing that endrin administered in the diet for up to 2 yr induced pituitary tumors in female but not in male Osborne-Mendel rats (NCI 1979b). The result for the Cochran-Armitage test for the incidence of adenomas of the pituitary in females was significant (p = .015) using the pooled controls, and the results of the Fisher exact test showed that the incidence in the high dose group was higher (p = .016) than that in the pooled controls. However, when the combined incidence of adenomas, carcinomas, adenocarcinomas, and chromophobe adenomas of the pituitary in female rats was compared with pooled controls, the results were not significant. In addition, two other studies did not report an increased incidence of pituitary tumors in Osborne-Mendel rats fed endrin for up to the 116 wk (Deichmann et al. 1970; Reuber 1978a). No pituitary tumors were reported in male or female B6C3F1 mice fed endrin for up to 2 yr (NCI 1979b).break

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Page 236 There was no evidence that endrin increased the incidence of tumors of the endometrium/uterus, ovaries, testicles, or prostate gland compared with controls. Lindane Bioassays of lindane have been conducted in two strains of rat (Osborne-Mendel and Wistar) (Fitzhugh et al. 1950; Ito et al. 1975; NCI 1977) and five strains of mouse (B6C3F1, dd, CF1, IRC-JCL, and NMRI) (Goto et al. 1972: Hanada et al. 1973; Thorpe and Walker 1973; Herbst et al. 1975; NCI 1977). None of the studies involved prenatal exposure to lindane. Overall, there was no evidence that lindane increased the incidence of tumors of the endometrium/ uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands compared with controls. However, there is some evidence that lindane might induce tumors of the liver, which led IARC (1987) to classify it as possibly carcinogenic to humans. Methoxychlor Bioassays of methoxychlor were conducted in Osborne-Mendel rats (Radomski et al. 1965; Deichmann et al. 1967; NCI 1978a), an unspecified species of rat (Hodge et al. 1952), and B6C3F1 mice (NCI 1978a). None of the studies involved prenatal exposure to methoxychlor. Overall, there was no evidence that methoxychlor increased the incidence of tumors of the endometrium/ uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands compared with controls. PCBs Bioassays of PCBs have been conducted in four strains of rat (Fischer 344, Sherman, Sprague-Dawley, and Wistar) (Kimbrough et al. 1975; NCI 1978d; Schaeffer et al. 1984; Norback and Weltman 1985). None of these studies involved perinatal exposure to PCBs. There was no evidence in any of the studies that PCBs induced tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, thyroid gland, pituitary gland, or adrenal glands. However, there is some evidence that PCBs induce cancers of the liver, which led IARC to classify it as probably carcinogenic to humans (IARC 1987). TCDD TCDD has been evaluated for carcinogenicity in Sprague-Dawley rats (Kociba et al. 1978; Brown et al. 1998), Osborne-Mendel rats (NTP 1982a), and B6C3F1 mice (NTP 1982a). Of the reproductive and endocrine organs considered by thecontinue

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Page 237 committee, an increased incidence of tumors from adult exposure to TCDD was observed only in the thyroid gland. There was an increase in the incidence of thyroid tumors in male and female Osborne-Mendel rats and in female B6C3F1 mice. The incidence of thyroid tumors was significantly increased (p = 0.001) in male rats and slightly increased in female rats administered 10 ppb TCDD by gavage twice a week for 2 yr (NTP 1982a). A significant increase (p = 0.009) in thyroid tumors was found in female, but not male, mice administered 0.3 ppb TCDD by gavage twice a week for 2 yr. However, there was no evidence that TCDD induced thyroid tumors in Sprague-Dawley rats fed up to 2 ppb TCDD in the diet for 2 yr (Kociba et al. 1978). One study was conducted to investigate the effects of prenatal exposure to TCDD on chemically induced carcinogenesis (Brown et al. 1998). Eight pregnant rats were gavaged with 1 µg/kg TCDD on d 15 post-conception. Female offspring were subsequently treated with dimethylbenzanthracene (DMBA) at 50 d of age. Ninety percent of the animals developed mammary adenocarcinomas compared with 79% of control animals that were exposed to TCDD during gestation. There was no evidence that adult exposure to TCDD caused tumors of the endometrium/uterus, ovaries, testicles, prostate gland, mammary gland, pituitary gland, or adrenal glands. In fact, a significant decrease in pituitary and adrenal gland tumors was observed in Sprague-Dawley rats (Kociba et al. 1978). The committee restricted its evaluation of carcinogenicity to selected reproductive and endocrine organs, but TCDD has been shown to induce tumors in laboratory animals in other organs, including the liver, lungs, thymus, hard palate, nasal turbinates, and skin. IARC (1997) concluded there is sufficient evidence in experimental animals that TCDD is carcinogenic, and classified TCDD as a human carcinogen. Experimental evidence suggests that the carcinogenic effects of TCDD are mediated through the Ah receptor which is a nongenotoxic, epigenetic mechanism (Ahlborg et al. 1995). Toxaphene Toxaphene was tested in bioassays using Osborne-Mendel rats and B6C3F1 mice (NCI 1979a). The tests involved exposure to adult animals only. There was some evidence that toxaphene induced thyroid tumors in rats. In male rats, the incidence of thyroid follicular-cell adenoma or carcinoma was 17% in the low-dose group, 25% in the high-dose group, and 4.5% in the control group. This increase was dose related (p = 0.007) when compared with pooled control data. In female rats, the incidence of thyroid follicular-cell carcinoma was 2% in the low-dose group, 17% in the high-dose group, and 2% in the control group. This increase was dose related using either matched (p = 0.022) or pooled (p = 0.008) control data. It is not known whether toxaphene induced the thyroid tumors through a hormonally mediated mechanism, although there is evidence that natu-soft

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Page 238 ral estrogens and DES induce follicular-cell adenoma in mice (Greenman et al. 1990). In the bioassay of toxaphene with mice, there was no evidence of an increased incidence of thyroid tumors. There was no evidence that toxaphene induced tumors of the endometrium/ uterus, ovaries, testicles, prostate gland, mammary gland, pituitary gland, or adrenal glands. However, there is some evidence that toxaphene might induce cancers of the liver, as well as the thyroid gland, which led IARC to classify it as possibly carcinogenic to humans (IARC 1987). Strengths and Limitations of the Cancer Bioassays Several factors must be taken into consideration when interpreting the results of cancer bioassays. The factors include the genetic background of animals, the appropriateness of the animal model, the dose and route of administration, and the timing and duration of exposure during an animal's life span. In addition, the data themselves can be subject to different interpretations. The factors will influence the certainty expressed in conclusions drawn from the underlying data base. Genetics The genetic background of an animal strain can influence the results of a study. For example, when Fisher, Wistar-Furth, and Sprague-Dawley rats were administered the chemical carcinogens 7,12-dimethylbenzanthracene (DMBA) or nitroso-methylurea, more than 80% of the rats developed mammary tumors (Daniel and Joyce 1984; el Abed et al. 1987; Kort et al. 1987; Thompson et al. 1995; Russo and Russo 1996). Other rats, such as Copenhagen, had a significantly lower incidence of tumor development. Different strains of rat and mice can also have varying incidences of spontaneous tumor formation that must be considered in the evaluation of carcinogenic agents. F344 rats are typically used in NCI bioassays because they have a low spontaneous incidence of mammary tumors, and B6C3F1 mice are used because they have a median incidence of liver tumors compared with other strains of mice (Eugene McConnell, Raleigh, N.C., personal commun., 1997). Many of the NCI bioassays reviewed used Osborne-Mendel rats, and no explanation was given for choosing this strain over F344 rats. In cases where the spontaneous incidence of tumors in the control animals is high, detecting an increase in the frequency of tumor formation in treated animals would require larger sample sizes than typically found in animal bioassays, which normally employ 20-25 animals per group. For example, a high incidence of pituitary tumors was found in the control animals in studies of lindane (NCI 1977), methoxychlor (NCI 1978a), endosulfan (NCI 1978b), DDT (NCI 1978c), and DDE (NCI 1978c). Although the incidence of those tumors in experimental animals was not increased in comparison with the controls, the high incidence ofcontinue

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Page 239 spontaneous pituitary tumors in the controls raises concern about the testing conditions. In addition, it should be noted that tumor incidence can be altered by dietary modulation. For example, it has been shown that reducing calorie intake increases survival, decreases the incidence of spontaneous tumors, and might alter susceptibility to chemical carcinogens in laboratory animals (NRC 1996a). Thus. the sensitivity of rodent bioassays might be altered by the administered diet. Animal Model In the NCI bioassays, both rats and mice were typically tested because of the belief that a given event occurring in both species could be expected to occur in a third species (for example, humans) (Weisburger 1983). With the exception of PCBs, all the HAAs reviewed by the committee were tested in rats and mice; PCBs were tested in rats only. However, the bioassays described in this chapter may or may not have used the most susceptible species or strains of animals. For example, the spontaneous development of prostate cancer is a rare event in most nonhuman species. Only a few rat strains, such as Noble and Lobund-Wistar, have been used as models of prostate cancer. In those rat strains, androgens and estrogens increase the incidence of prostate cancer. The Noble rat appears to be uniquely sensitive to sex hormones for prostate cancer induction (Ofner et al. 1992). In this model, dysplasia in the dorsolateral lobe of rats treated with testosterone and estradiol is almost identical to the premalignant lesions described in the human gland (McNeal and Bostwick 1986). None of the studies on environmental HAAs described in this chapter were conducted using the Noble or Lorbund-Wistar strains. Also, there are no adequate animal models to test for endometrial or germinal-cell testicular cancers (which are the type of testicular tumor that is increasing in certain human populations). Dose and Route of Administration In NCI studies, animals are exposed via the most likely route of exposure in humans. Test animals are typically administered the maximum tolerated dose (MTD) and one-half of the MTD in cancer bioassays. The MTD is defined as the highest dose that does not alter the test animal's longevity or well-being because of noncancer effects (NRC 1993). Thus, the doses are not designed to be representative of environmental exposures. In the NCI (1978b) study on endosulfan, the sublethal doses administered to the male animals caused severe secondary effects and early death; therefore, carcinogenicity could not be assessed adequately. Because the highest dose used in a chronic toxicity study is based on the results from a prechronic toxicity study, the MTD is exceeded in some cases, as in the NCI endosulfan study. The other NCI and NTP studies discussed in this chapter showed no indication that the MTD was exceeded. However, there arecontinue

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Page 240 concerns about the design of the typical long-term rodent bioassay, including the use of the MTD (see NRC 1993). Timing and Duration of Exposure The duration of exposure and period of observation must be considered when interpreting the results of a cancer bioassay. Experimental animals should be exposed to a range of doses of an agent for up to the life span of the animal to detect the effects of carcinogens with long latency periods and with multiple modes of action (for example, initiation or promotion). It is also important to take into account the stage in an animal's life cycle that exposure occurs. DES has been shown to cause cancer in humans and animals (Newbold 1995) when exposure occurs during critical periods of development but not when exposure occurs after the critical period. The timing of exposure during postnatal life also could affect carcinogenicity. In DMBA-induced mammary cancer in rats, a ''window of vulnerability" was identified between d 45 and 55 of life (Russo and Russo 1978); the administration of the carcinogen during that period significantly increased the incidence of carcinoma and decreased the latency period. The effect is explained by the induction of intense proliferative activity of structures called terminal end buds, from which new gland ducts originate during the period (Russo and Russo 1987). Perinatal exposure was not addressed in the bioassays described in this chapter with the exception of several bioassays in which mice were exposed to DDT during fetal life, lactation, and after weaning by feeding or gavage for up to 6 mo or their life span. Those studies produced negative findings. In a recent report, the U.S. Environmental Protection Agency's (EPA) FIFRA/Scientific Advisory Panel (SAP) reviewed an EPA analysis of combined perinatal and adult exposure, perinatal only exposure, and adult only exposure carcinogenesis bioassays to determine if the age of initial exposure to a chemical influences the carcinogenic response (EPA 1997). In the analysis, 69 carcinogenesis bioassays were reviewed. The studies analyzed included six NTP bioassays (Chhabra et al. 1992, 1993a,b), 13 unpublished FDA bioassays, and other studies previously reviewed by McConnell (1992). Chemicals tested in the bioassays reviewed by McConnell (1992) include three HAAs (dieldrin. DDT, and TCDD). The SAP agreed with the EPA's conclusion that perinatal exposure rarely identifies carcinogens that are not found in standard (adult only) carcinogenesis bioassays, and combined perinatal and adult exposure slightly increases the incidence of a given type of tumor. With respect to the latter, it is not known if this reflects the effect of an increased length of exposure, a heightened sensitivity of the young animal to the carcinogenic effects of the chemical, or variability in the experimental design or results. EPA noted the available data for drawing conclusions are not very robust and has developed criteria for the inclusion of perinatal exposure into the standardcontinue

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Page 241 carcinogenesis bioassay. The criteria include factors such as the likelihood of widespread exposure to women of childbearing age, infants and children, and specific toxicity to the developing fetus. A weight-of-the-evidence approach is applied to these factors to determine if a chemical is a candidate for perinatal carcinogenesis testing. The Food and Drug Administration (FDA) (1982) has also developed a set of criteria for determining candidates for perinatal carcinogenesis bioassays that are based on use, exposure, and toxicity seen in developmental toxicity and reproductive studies. Other Interpretations One investigator has published different interpretations of some of the NCI and FDA studies described in this chapter (see Table 8- 1 footnotes b, c, d, and e). Reuber (1978b, 1979, 1980, 1981) reported positive results in reanalyses of NCI (1976, 1977, 1978a,b) studies and unpublished FDA studies, all of which reported negative results. Reuber concluded that methoxychlor increased the incidence of mammary, ovarian, testicular, pituitary, thyroid, and adrenal gland tumors, that endosulfan increased the incidence of all tumors of the female genital tract, and lindane increased the incidence of ovarian, pituitary, thyroid and adrenal gland tumors, and that chlordecone increased the incidence of pituitary tumors. Because Reuber (1978b, 1979, 1980, 1981) did not indicate why he considered the original NCI (1976, 1977, 1978a,b) interpretation of the tissues questionable or how the tissues were reexamined, his reanalysis cannot be independently verified. Summary and Conclusions Given that there are data from animal and human studies that indicate endogenous estrogens play some role in increasing the incidence of tumors in various endocrine glands in humans (Key and Pike 1988; Brinton and Hoover 1993; Nandi et al. 1995), and animals (Sonnenschein et al. 1974; Wiklund and Gorski 1982; Greenman et al. 1990; Nandi et al. 1995) and that the introduction of HAAs into the environment has preceded and overlapped the increasing incidence rates of some types of cancer, an association between HAAs and cancer is a reasonable hypothesis. Animal bioassays have been conducted to assess the carcinogenicity of aldrin and dieldrin, bisphenol A, BBP, chlordecone, DDD, DDE, DDT, endosulfan, endrin, lindane, methoxychlor, PCBs, TCDD, and toxaphene. Overall, the available animal data do not support an association between environmental HAAs and cancers of the female and male reproductive systems and endocrine organs. However, some of the HAAs evaluated have been shown to induce tumors in other organs. IARC has classified lindane, toxaphene, p,p'-DDT, and chlordecone as possibly carcinogenic, PCBs as probably carcinogenic, and TCDD as carcinogenic in humans.break

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Page 242 The few human studies that are available are in general agreement with the animal studies described in this chapter. Human studies conducted to date do not find an association between breast, endometrial, or testicular cancers and DDT. DDE, PCBs, or TCDD (see Chapter 9). However, several of these studies are limited by factors such as small case numbers and lack of exposure measurements in body fluids and tissues. Based on the available data, animal studies have also not found an association between DDT, DDE, PCBs, or TCDD and breast, endometrial, or testicular tumors. Liver cancer in wild fish populations has been conclusively linked to the presence of known carcinogens, especially polynuclear aromatic hydrocarbons (PAHs), in the environment (Harshbarger and Clark 1990; Baumann and Harshbarger 1995). The liver is an estrogen-responsive organ in fish, and laboratory experiments in two-stage models of carcinogenesis in two species have indicated that estradiol is a tumor promoter in some fish (Nunez et al. 1989; Cooke and Hinton 1999). It is possible that environmental estrogens act in this way in wild fish, but it has not been confirmed. Tumors in endocrine organs of fish occur in regions contaminated with pesticides: but it is not known whether those chemicals are the causative agents (Harshbarger and Clark 1990). Body burdens of PCBs, DDT, and DDE have been decreasing over the past 30 yr largely due to regulatory intervention. However, the body burden and exposure to the majority of environmental HAAs, including other estrogenic pesticides, antioxidants, and plasticizers is not known, and the potential cumulative effects of environmental HAAs is yet to be fully explored. In addition, not all HAAs have yet been tested for carcinogenicity, including some phthalates and alkyl phenols. The large number of studies reviewed by the committee that examined the carcinogenicity of some of the most studied HAAs in many species under a great variety of experimental circumstances failed to adduce compelling evidence that HAAs induce cancers of the female and male reproductive systems and other endocrine organs. However, most of the available studies involved high-dose, postnatal testing. Little or no data are available on whether prenatal exposure to HAAs can cause cancers of the endocrine system. Recommendations Because perinatal exposure for the most part has not been addressed with respect to carcinogenesis, research in laboratory animals is needed on the role of prenatal exposure to suspected chemicals in inducing cancers later in life or in subsequent generations. Initial studies should focus on HAAs that have been shown to induce cancer of the thyroid, pituitary, and adrenal glands in some laboratory animals.break