Ethinyl estradiol is a potent synthetic estrogen widely used in pharmaceutical preparations. Its high potency and widespread use led to its selection by the National Toxicology Program for inclusion in studies to examine endocrine disrupting compounds with estrogenic activity both because of its utility as a positive control to which weaker estrogens can be compared and because of potential human developmental exposures resulting from unintentional continuation of the use of oral contraceptives containing ethinyl estradiol during early pregnancy. The study protocol utilized Sprague-Dawley rats and was designed to evaluate the effects of short-term, multigenerational, and long-term exposures to doses of estrogenic agents that produce subtle reproductive tract lesions in developmentally exposed Sprague-Dawley rat pups.
Results of the 2-year study are reported in this Technical Report, and results of short-term reproductive dose range-finding and multigenerational reproductive toxicology studies are reported separately (NTP TR 547). Data from the short term reproductive dose range finding study were used to select dietary exposure concentrations of 0, 2, 10, and 50 ppb for the current study. The multigenerational reproductive toxicology study examined F0 through F4 generations with F5 litters terminated at weaning and focused on reproductive endpoints. Animals were exposed from the time that the F0 generation was 6 weeks old through weaning of the F3 generation, and animals of the F0 through F4 generations were necropsied at 20 weeks of age.
The current study was a 2-year dietary study utilizing three exposure arms: continuous exposure from conception through 2 years (designated F1 continuous, or F1C), exposure from conception through 20 weeks followed by control diet to 2 years (designated F1 truncated at postnatal day (PND) 140, or F1T140), and exposure from conception through weaning followed by control diet to 2 years (designated F3 truncated at PND 21, or F3T21). The "F3" designation for the F3T21 arm indicates that these animals were siblings of the F3 animals from the multigenerational reproductive toxicology study. The F1C and F1T140 animals were also siblings, but were derived from a separate breeding that was identical to the procedure used to produce the F1 generation of the multigenerational reproductive toxicology study. The animals in this study were exposed to ethinyl estradiol during various phases of their lives from conception until termination at 2 years, and the ingested doses varied over the course of the study. During pregnancy, the ingested doses of the dams were approximately 0, 0.2, 0.9, or 5.8 µg/kg per day. During lactation, the dams' ingested doses were 0, 0.3, 2.0, or 10.3 µg/kg per day. The mean directly ingested ethinyl estradiol doses during the period prior to PND 140 were approximately 0.2, 0.9, or 4.9 µg/kg per day for females and 0.2, 0.8, or 4.5 µg/kg per day for males. For the period between PND 140 and the end of the study, mean ingested doses were approximately 0.1, 0.6, or 3.3 µg/kg per day for females and 0.1, 0.4, or 2.1 µg/kg per day for males. Under these dosing conditions, serum levels in the high dose group (50 ppb) were below the limit of detection (10 pg/mL) of a sensitive liquid chromatography-mass spectrometry method (Twaddle et al., 2003).
For the current study, 50 animals per sex were initially assigned to each exposure group in each arm of the study. In control groups, histopathology data from one or two additional animals that had been assigned as sentinels but that became moribund or died early were also included in the analysis and presentation. Survival was similar in all control and exposed groups and ranged from 55% to 70% for males and 32% to 58% for females. The mean body weights of of the 2 and 50 ppb F1C males were less than those of the controls throughout the study, and the mean body weights of the 2 and 50 ppb F1T140 males were less than those of the controls early in the study. The mean body weights of all exposed groups of F1C and F1T140 females were less than those of the controls throughout the study.
In males, the only neoplastic effects observed were positive trends in the incidences of preputial gland epithelial neoplasms and mammary gland adenoma or adenocarcinoma (combined) in F3T21 animals. There were increased incidences of mammary gland alveolar hyperplasia in the 10 and 50 ppb F1C and F1T140 groups and in the 50 ppb F3T21 group. There were increased incidences of ductal hyperplasia in the mammary gland of 50 ppb F1C and F1T140 males. In the liver, there were increased incidences of basophilic and eosinophilic foci in 50 ppb F1C and F1T140 males.
In females, there was a marginally positive dose trend in the incidences of uterine stromal polyps in F1T140 animals and a significantly increased incidence of uterine stromal polyps in 2 ppb F3T21 animals. In the F1C females, increased incidences of uterine nonneoplastic lesions included endometrial hyperplasia in the 50 ppb group, squamous metaplasia in the 10 and 50 ppb groups, and atypical focal hyperplasia in all exposed groups. In the uterus of F3T21 females, there were increased incidences of atypical focal hyperplasia in all exposed groups and an increased incidence of squamous metaplasia in the 50 ppb group. There was an increased incidence of eosinophilic focus in the liver of 50 ppb F1C females and an increased incidence of basophilic focus of the liver in the 50 ppb group of F1T140 females. There was no discernible effect of ethinyl estradiol on the time of onset of aberrant estrous cycles in any arm of the study.
Under the conditions of this 2-year feed study with continuous exposure to the test compound from conception through termination (F1C), there was of carcinogenic activity no evidence of ethinyl estradiol in male or female Sprague-Dawley rats exposed to 2, 10, or 50 ppb. Nonneoplastic lesions were observed in the mammary gland and liver of males and in the uterus and liver of females.
Under the conditions of this 2-year feed study with exposure to the test compound from conception through 20 weeks followed by control feed until termination (F1T140), there was no evidence of carcinogenic activity of ethinyl estradiol in male Sprague-Dawley rats exposed to 2, 10, or 50 ppb. There was equivocal evidence of carcinogenic activity of ethinyl estradiol in female Sprague-Dawley rats based on marginally increased incidences of uterine stromal polyps. Nonneoplastic lesions were observed in the mammary gland and liver of males and in the liver and clitoral gland of females.
Under the conditions of this study where offspring of two prior generations of animals exposed to ethinyl estradiol in feed were exposed from conception through weaning (PND 21), followed by control feed through termination (F3T21), there was equivocal evidence of carcinogenic activity of ethinyl estradiol in male Sprague-Dawley rats based on increased incidences of preputial gland epithelial neoplasms and a marginal increased incidence of mammary gland adenoma or adenocarcinoma (combined). A significantly increased incidence of male mammary gland alveolar hyperplasia was also observed. There was equivocal evidence of carcinogenic activity of ethinyl estradiol in female Sprague-Dawley rats based on marginally increased incidences of uterine stromal polyps. Increased incidences of squamous metaplasia and atypical hyperplasia in the uterus and hyperplasia in the clitoral gland were also observed.