Two-generation reproductive toxicity studies using rats of benzophenone, n-butylbenzene, butyl benzyl phthalate, 2,4-dichlorophenol, dicychlohexyl phthalate, diethyl phthalate, 4-nitrotoluene, lindane and vinclozolin, were performed to investigate whether these chemicals have endocrine-mediated effects with the support of the Ministry of Economy, Trade and Industry and the Ministry of the Environment. Benzophenone exposure was via the diet at concentrations of 0, 100, 450 or 2000 ppm, n-butylbenzene was administered orally by gavage at dose levels of 0, 30, 100 or 300mg/kg/day, butyl benzyl phthalate was administered orally by gavage at dose levels of 0, 100, 200, or 400 mg/kg/day, 2,4-dichlorophenol was administered in the diet at concentrations of 0, 500, 2000 or 8000 ppm, dicyclohexyl phthalate was given in the diet at concentrations of 0, 240, 1200 or 6000 ppm, diethyl phthalate was administered in the diet at concentrations of 0, 600, 3000 or 15000 ppm, 4-nitrotoluene was administered orally by gavage at doses of 0, 40, 80, or 160 mg/kg/day, lindane exposure was in the diet at concentrations of 0, 10, 60, or 300 ppm, and vinclozolin treatment was by feeding diet at concentrations of 0, 40, 200 or 1000 ppm.

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The reproductive toxicity of benzophenone (BZP) was evaluated in a two generation test in which male and female Sprague-Dawley (SD) rats, parental (F0) and first generation (F1), were exposed to BZP by feeding diet containing BZP at concentrations of 0 (control), 100, 450 or 2000 ppm. With regard to the effects of BZP on the F0 and F1 parental animals, inhibition of body weight gain and food consumption, significantly elevated renal weights, dilatation of the renal proximal tubules, and regeneration of the proximal tubular epithelium were recognized at doses of 450 ppm and 2000 ppm, along with an increase in hepatic weight and centrilobular hepatocytic hypertrophy, considered as vital adaptive changes, at the doses of 100 ppm or more. Obvious effects on the endocrine system and reproductive toxicological effects were not found even at the dose of 2000 ppm in the F0 or F1 parent. There were no test substance related changes in the estrous cycle, reproductive capability, delivery and lactation, sperm parameters, serum hormone levels, or necropsy findings. As for effects on the offspring, inhibition of body weight gain was observed in both the F1 and F2 males and females of the 2000 ppm group. No effects of BZP treatment were recognized in the number of male and female F1 or F2 pups delivered, viability, anogenital distance (AGD), physical development, the results of reflex and response tests, or on the observation results of external abnormalities. From the present study of BZP administered to rats over two successive generations, the no observed effect level (NOEL) on the parental animals is concluded to be less than 100 ppm. Concerning the effects on the endocrine system and the reproductive toxicity in the parental animals, the NOEL is 2000 ppm. In terms of the effects on the offspring, the NOEL is considered to be 450 ppm.

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N-butylbenzene (n-BB), which is suspected of having endocrine disrupting effects, was administered orally by gavage at dose levels of 0, 30, 100, and 300 mg/kg/day to groups of Crj: CD (SD) IGS rats (24 males and 24 females per group) over 2 generations, and the effects on fertility of the parental animals and development/growth of the offspring were investigated. In the F0 and F1 parental animals, n-BB at the doses of 30 mg/kg/day and above increased the liver weights, and the doses of 100 and 300 mg/kg/day increased the kidney weights and caused histopathological changes in the liver and kidney. Moreover, the dose of 300 mg/kg/day also increase adrenal gland weights and there was a tendency for inhibition of body weight gain. With respect to effects on fertility, no significant findings were noted in the F0 parental males and females. Furthermore, it was concluded that n-BB did not induce serious reproductive toxicity in the F1 parental animals and no effects on the endocrine system were recognized. In the F1 and F2 offspring, n-BB at 300 mg/kg/day increased the thymus weights.

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A two-generation reproductive toxicity study with extra parameters was performed for Butyl benzyl phthalate (BBP). The compound was administered orally by gavage with the doses of 0, 100, 200, or 400 mg/kg/day to groups of 24 Crj:CD^®(SD)IGS rats of both sexes to confirm the utility of the protocol for identification of non-steroid chemicals with endocrine activity by ssessing effects on parental animals and offspring. Softening of the testes, diffuse atrophy of testicular seminiferous tubules, decreased spermatozoa and/or residual germ cells in the epididymal lumina were observed in the F1generation after doses more than 100 mg/kg, lowering of the F1 epididymal weights at doses more than 200 mg/kg, along with low F0 epididymal weights, Leydig cell hyperplasia, residual germ cells in the epidimymal lumina, and low seminal vesicle weights, small testes and epididymes, partial aplasia or aplasia of the epididymes, and Leydig cell hyperplasia in the F1 generation with 400 mg/kg. With regard to effects on the reproductive capacity, F1 parents at the dose of 400mg/kg showed a reduced fertility index and delayed preputial separation of the penis. In the offspring, lowered body weights in the F1 case, and change in anogenital distance in the F1 females and F2 males were observed at doses more than 100 mg/kg, with low splenic weights at 400 mg/kg in both generations. Thus, the utility of this protocol was confirmed. In the parental animals, the no observed effect level (NOEL) and the no observed adverse effect level (NOAEL) were less than 100 mg/kg/day, and no serious effects on the reproductive capacity were induced at doses less than 200 mg/kg/day. The NOEL and NOAEL for the growth and development of offspring were concluded to be less than 100 mg/kg/day.

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- A two-generation reproductive toxicity study was conducted with 2,4-dichlorophenol (2,4-DCP), an agent suspected of exerting endocrine disrupting effects. Wistar-Hannover rats, 24/sex/group, were given diet containing 2,4-DCP at dose levels of 0, 500, 2000 or 8000 ppm to examine the potential effects of the test substance on parental animals and their offspring over 2 successive generations. Neither clear systemic nor reproductive toxicity of 2,4-DCP was apparent in the 500 ppm group. In the 2000 ppm group, mean body weight gain and food consumption of females were lowered significantly during the treatment period. Effects on body weights and food consumption were more serious in the 8000 ppm group, both males and females being significantly affected. Reproductive effects of the test substance were also observed in the 2000 and 8000 ppm groups dose-dependently. Observations included significantly increased uterine weights of F1 and/or F2 female weanlings and reduced numbers of implantation sites and live births of F1 parental females. These results suggest that 2,4-DCP has weak reproductive toxicity, possibly based on endocrine activity. However, the basic mechanisms for apparent estrogenic effects of 2,4-DCP remain to be elucidated.

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The reproductive toxicity of dicyclohexyl phthalate (DCHP) was evaluated in a two generation test in which male and female Sprague-Dawley (SD) rats of parental (F0) and F1 generation were exposed to DCHP in the diet at concentrations of 0 (control), 240, 1200 or 6000 ppm. With regard to the effects on the F0 and F1 parental animals, changes included inhibition of body weight gain and food consumption, diffuse hypertrophy of hepatocytes, and hypertrophy of thyroidal follicular epithelial cells at the doses of 1200 ppm and 6000 ppm. The following changes were observed in the 6000 ppm group: increase weights of the liver and thyroid, increased hyaline droplets in the renal proximal tubular epithelium (F0 and F1 males), reduction of prostatic weight (F1 males), and diffuse and/or focal atrophy of testicular seminiferous tubules (F1 males). In addition, slight prolongation of the estrous cycle was noted in the F0 females of the 6000 ppm group, along with reduced spermatid head counts in the testes (homogenation-resistant spermatids) in F1 male receiving doses of 1200 ppm or 6000 ppm. It is thought that the prolonged estrous cycle was secondary to the suppression of body weight gain. There were no test substance related changes in clinical signs and reproductive capability (mating, fertility, gestation and birth index), or in data for the delivery and lactational periods, or serum hormone levels. With regard to effects on the offspring, inhibition of body weight gain was found in the F1 and F2 6000 ppm, and decrease of anogenital distance (AGD) and appearance of areola mammae were observed in the F1 male 6000 ppm and F2 male receiving doses of 1200 ppm or 6000 ppm. No effects of DCHP treatment on the offspring were observed on results of clinical signs, the number of the pups delivered, sex ratio, viability, physical development, reflex and response tests, external abnormalities, organ weights, or necropsy findings. From the present study of DCHP administered to rats over two-generations, the no observed effect level (NOEL) for effects on the parental animals including the endocrine system, is considered to be 240 ppm. With regard to the reproductive toxicological effects on the parental animals, the NOEL is 240 ppm for males and 1200 ppm for females. For offspring, the NOEL values are concluded to be 240 ppm for males and 1200 ppm for females.

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A two-generation reproductive toxicity study was performed to evaluate the effects of diethyl phthalate on parental reproductive performance, including features of the endocrine system and development and growth of offspring at dietary dose levels of 0, 600, 3000 and 15000 ppm. In F0 and F1 parents, no treatment-related adverse effects were observed in clinical findings, body weights, food consumption, reproductive parameters, and gross or histopathological findings in any treated group. Increased liver weights and enhanced activities of metabolic enzymes were observed in F0 males at 15000 ppm. F0 males also exhibited an increase in the content of CYP3A2, a cytochrome P450 isozyme, at 15000 ppm, and a decrease in the levels of serum testosterone at 3000 and 15000 ppm, suggesting sex steroid metabolism might be changed. However, these were not considered adverse effects because the degree of change was too slight to affect the reproductive capability to produce progeny. Body weight gains before weaning were inhibited in F1 and F2 pups and vaginal opening was slightly delayed in F1 females at 15000 ppm. No changes were observed in the reproductive performance. Therefore, the no-observed-adverse-effect level (NOAEL) from this study is considered to be 15000 ppm for parental animals, and 3000 ppm for development and growth of the pups.

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4-nitrotoluene (4-NT) was administered orally at doses of 0, 40, 80, or 160 mg/kg/day by gavage to 24 Crj:CD^®(SD)IGS rats of each sex per group over two successive generations, and the effects on reproductive capacity in the parental animals and growth and development of the offspring were investigated. In the F0 and F1 parents, increased hepatic and/or renal weights were observed at the doses of 40 mg/kg or more in both generations, with lowered body weights in the F1 case and reduced feeding efficiency, histopathological changes in the kidney and spleen at doses of 80 and 160 mg/kg, as well as worsening of clinical signs and death during the perinatal period at 160 mg/kg in both generations. With regard to effects on the reproductive capacity, reduced vaginal opening was observed at 160 mg/kg in the F1 generation. However, no abnormalities were observed in the endocrine and reproductive organs or in serum hormone levels. In the F1and F2 offspring, decrease in body weight gain and brain weights was observed at the doses of 80 and 160 mg/kg and reduced viability, with elongation or a tendency for elongation of the male anogenital distance at 160 mg/kg. Thus, the possibility that 4-NT exerted endocrine disrupting effects seems to be very low under the conditions of this study, and when the substance was administered to rats over two generations, doses less than 160 mg/kg/day did not induce any marked adverse effects on the reproductive capacity in the parental animals, with the no observed effect level (NOEL) and the no observed adverse effect (NOAEL) on growth and development in the offspring concluded to be 40 mg/kg/day.

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A two-generation reproduction toxicity study in rats adding extra endpoints to detect endocrine disrupting activity was conducted using lindane by dietary administration at 0, 10, 60, and 300 ppm, for investigation of its utility. The extra endpoints included anogenital distance (AGD), nipple development, sexual maturation (vaginal opening and preputial separation), estrous cycle, spermatogenesis, sex organ weights, and blood hormone concentrations (thyroid and sex hormones). F1 offspring were examined for emotionality (open field test), motor coordination (rotarod test), as well as learning and memory (pole-climbing test). Hepatic drug-metabolizing enzyme activities were also measured. The results revealed general toxicological effects on parental animals, influence on reproductive function, and altered development of offspring; however, they did not demonstrate any distinct changes in the extra endpoints for detection of endocrine disrupting activity. Adult toxicity was observed in both F0 and F1 animals, including suppressed body weight gain and reduced food consumption in both sexes, and deaths of females at 300 ppm. Convulsions and irritability were observed during the perinatal period in pregnant F1 females given 300 ppm. Pathological examination revealed increased liver weights and centrilobular hepatocellular hypertrophy in both sexes and generations at 10 or 60 ppm and above; in addition, increased kidney weights and increased hyaline droplets in the proximal tubule epithelium, and basophilic renal tubules in males were noted at 10 ppm and above. Pituitary weights were decreased in F0 females and in F1 males and females and adrenal weights were increased in F1 males and females at 300 ppm; however, no histological changes were observed, and manifestations suggesting endocrine disrupting activity related to these changes were lacking. Hypertrophy of the thyroid follicular epithelium in F0 females at 300 ppm and in F1 males at 60 and 300 ppm, and decreases in T3 and/or T4 in both sexes and generations at 300 ppm were presumed to be secondary changes associated with the induction of hepatic drug-metabolizing enzymes. Blood hormone analysis revealed no changes in sex hormones attributable to lindane in males or females. Hepatic drug-metabolizing enzyme activities were increased dose-dependently from 10 ppm in both sexes and generations, with the rise in BROD activity being the most prominent. There were also increases in MROD, EROD, T-6β-OH, and T4-UDP-GT activities (BROD >> EROD > MROD, T-6β-OH, T4-UDP-GT). This suggests that while lindane most strongly induces CYP2B, it also upregulates a number of other drug metabolizing enzymes, such as CYP1A, CYP3A, and UDP-GT. As for effects on reproductive function, lack of maternal behavior, including lactation and retrieval behavior, and consequent total litter loss were observed in F1 dams at 300 ppm. There were no effects of lindane on the estrous cycle, spermatogenesis, mating, fertility, pregnancy, or parturition. Neonatal toxicity was observed in both sexes and generations, including suppressed body weight gain at 60 and 300 ppm, and decreased thymus and spleen weights without histological change at 300 ppm. The postnatal survival rate in F2 offspring was decreased due to lack of maternal behavior in dams at 300 ppm.

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A two-generation reproduction toxicity study in rats adding extra endpoints to detect endocrine disrupting activity was conducted using vinclozolin by dietary administration at 0, 40, 200, and 1000 ppm, for investigation of its utility. The extra endpoints included anogenital distance (AGD), nipple development, sexual maturation (vaginal opening and preputial separation), estrous cycle, spermatogenesis, sex organ weights, and blood hormone concentrations (thyroid and sex hormones). Hepatic drug-metabolizing enzyme activities were also measured. The results revealed changes due to vinclozolin in the AGD, nipple development, sexual maturation, sex organ weights, and blood sex hormone concentrations in males of both parental animals and offspring, even at the lowest dose of 40 ppm, confirmed by results for the classical endpoints of histopathological examination at 200 ppm and mating at 1000 ppm. The effects on parental males included increased pituitary and testis weights, and decreased epididymis weights at 1000 ppm in both generations, and decreased prostate and epididymis weights at 200 and 1000 ppm and seminal vesicle weights at 1000 ppm in F1 males. Histopathological examination revealed hypertrophy of the basophilic cells in the pituitary at these two doses, and diffuse hyperplasia of the testicular interstitial cells and atrophy of the seminal vesicle mucosa at 1000 ppm in F0 and F1 males. In addition, F1 males demonstrated decrease in prostate fluid at 200 and 1000 ppm. Blood hormone analysis revealed increases in LH, FSH, testosterone, and DHT in F0 and F1 males at 1000 ppm. General toxicological effects included suppressed body weight gain in F0 and F1 females and in F1 males, and reduced food consumption in F0 and F1 females at 1000 ppm. Histopathological examination revealed centrilobular hepatocellular hypertrophy in males at 200 and 1000 ppm and in females at 1000 ppm, increased lipid droplets in the adrenal zona fasciculata and zona glomerulosa in males at 200 and 1000 ppm and in females at 40 ppm and above, and hyperplasia of ovarian interstitial cells and vacuolation of lutein cells in females at 1000 ppm in both generations. Almost all the tissue changes were accompanied by changes in weights. Decreases in T3 and/or T4 were observed in both sexes and generations at 1000 ppm and in F0 females at 200 ppm. However, these were presumed to be secondary to induction of hepatic drug-metabolizing enzymes, activities being increased for a range of enzymes in both sexes and generations at 1000 ppm. Rise in BROD activity was the most prominent, suggesting that vinclozolin mainly induces CYP2B. As for effects on reproductive function, a marked decrease in the fertility index caused by male infertility was observed in F1 animals at 1000 ppm. However, no effects on spermatogenesis were seen in either F0 or F1 males. Since cleft prepuce and penile hypoplasia were observed in infertile males, it is probable that the cause of infertility in F1 males was related to morphological abnormalities in the external genitalia. Vinclozolin did not affect the estrous cycle, mating, fertility, pregnancy, parturition, or nursing behavior in either F0 or F1 females. In offspring, in addition to suppressed body weight gain in F1 males and females at 1000 ppm, neonatal toxicity caused by antiandrogen activity of vinclozolin was observed in F1 and F2 males. Effects included shortened AGD in F1 males at 1000 ppm and in F2 males at 200 and 1000 ppm, and nipple/areola remnants in F1 males at 200 and 1000 ppm and in F2 males at 40 ppm and above. In addition, decreased epididymis weights at weaning and morphological abnormalities of the external genitalia, including cleft prepuce, penile hypoplasia, and vaginal pouch, were seen in F1 and F2 males at 1000 ppm.

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