Evaluation of the 20-day pubertal female assay in Sprague-Dawley rats treated with DES, tamoxifen, testosterone, and flutamide

The Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) has recommended the rodent pubertal female assay as a Tier I test to detect potential endocrine disrupters (EDs). This assay is designed to screen estrogenic activity in immature rats exposed to chemicals during sexual maturation. The aim of this study was to evaluate whether this assay can detect the EDs with effects brought about through various mechanisms. Immature Sprague-Dawley female rats (21 days of age) were dosed daily for 20 days by oral gavage (DES, tamoxifen, and flutamide) or sc injection (testosterone). The mean age at vaginal opening (VO) was 32.3 ± 0.5 days in control rats. Although VO was unaffected by DES at doses of 0.2 and 1.0 μg/kg, a high dose of DES (5.0 μg/kg) significantly advanced the age at VO to 24 days. Both tamoxifen (50 and 200 μg/kg) and flutamide (25 mg/ kg) also significantly accelerated VO to 27.8 ± 0.5, 25.1 ± 0.1, and 26.1 ± 0.1, respectively. However, testosterone dose-dependently delayed VO (exposure to 1.0 mg/kg extended VO to 37.3 ± 0.8 days, and VO did not occur in 2 of 10 animals by the time of necropsy at 41 days of age). Estrous cyclicity was monitored in rats from VO to necropsy. Irregular cycles were observed in the groups treated with DES (5.0 μg/kg), tamoxifen (200 μg/kg), testosterone (1.0 mg/kg), and flutamide (25 mg/kg). High dose of DES showed a persistent estrus state throughout the entire observation period. In addition, the number of days in diestrus was increased by tamoxifen (200 μg/kg) and flutamide (25 mg/kg) treatments. Significant decreases in ovarian weight were observed in 5.0 μg/kg DES (64% of control), 25 mg/kg flutamide (76% of control), and 200 μg/kg tamoxifen (47% of control). Testosterone also significantly decreased the ovarian weights in all treatment groups. Uterine weights were also decreased significantly at high doses of tamoxifen (200 μg/kg, 39% of control) or testosterone (1.0 mg/kg, 47% of control). In hormone analysis, tamoxifen significantly increased serum E₂ levels at 50 μg/kg. The mean serum levels of TSH were significantly increased in tamoxifen (10 and 50 μg/kg), testosterone (0.2 mg/kg), and flutamide (1.0 and 25 mg/kg) treatment groups compared with the control. However, serum T₄ levels were significantly reduced by testosterone. Furthermore, serum T₃ levels were significantly increased in DES, tamoxifen (10 and 50 μg/kg), testosterone (1.0 mg/kg), and flutamide (1.0 and 5 mg/kg). Our data demonstrate that the rodent pubertal female assay is useful for identifying potential EDs having not only estrogenic/ antiestrogenic but also androgenic/antiandrogenic activities. However, further validation study is necessary to identify chemicals that operate through other action mechanisms, including steroid biosynthesis inhibitors and thyroid inhibitors. Moreover, additional data on other compounds with weak endocrine disrupting activity will be required to further characterize the sensitivity of the female pubertal assay.