This study evaluated the potential for diethylstilbestrol to alter the skeletal morphology of term mouse fetuses when administered to a limited number of pregnant animals. DES was administered during a gestational period corresponding to the period of known human fetal exposure. Developmental DES exposure has been shown to alter subsequent bone deposition in female mice during adulthood at doses below those causing reproductive tract anomalies. The present study was designed to determine if alterations in bone development or other developmental effects could be detected in prenatally exposed mouse fetuses using methods for assessment of developmental toxicity. In addition, the methods for staining and preserving fetal skeletons in this study allow subsequent planned in-house assessments of skeletal development by the sponsor using alternate approaches (e.g., morphometric evaluation or more detailed assessment of skeletal ossification patterns).
DES (CAS No. 56-53-1 ) was administered subcutaneously in corn oil to mated Swiss (CD-1) mice (12/group) on gd 9 through 16 at levels of 0, 2.5, 5, 10, 50, or 100 mg/kg/day. A trioctanoin vehicle control group and 100 mg/kg/day DES in trioctanoin group (12/group) were dosed concurrently to provide comparison of data using a different vehicle. Animals were observed daily for clinical signs of toxicity on gd 9-17. Food and water weights were recorded for the animals in each group on gd 0, 3, 6, 9, 12, 16, and 17. Body weights were taken on gd 0, 3, 6, and 9-17. All animals in the developmental toxicity study were killed on gd 17 and examined for maternal body and organ weights, implant status, fetal weight, sex, and morphological development (external, visceral and skeletal).
For animals dosed with DES in corn oil, no maternal lethality was observed. Pregnancy rates were 100%, 100%, 100%, 83%, 100%, and 70% for the control through high dose groups. maternal body weight gain was decreased in the 50 and 100 mg/kg/day groups compared to the corn oil controls during the gestational period (gd 0-17), and in the high dose compared to the com oil controls during the treatment period (gd 9-16). Corrected maternal body weight gain was decreased in all the DES in corn oil-treated groups compared to the corn oil control group. No dose-related clinical signs were observed. maternal relative food and water consumption for the DES in com oil groups displayed little effect of treatment. At necropsy, relative maternal liver weight was increased in the high dose group, and gravid uterine weight was decreased. The number of implantation sites per dam exhibited a decreasing trend; the number of corpora lutea per dam was decreased at greater than 10 mg/kg/day. The percent resorptions per litter was increased 3, 4, and 5-fold in the 10, 50, and 100 mg/kg/day groups, respectively. Average live litter size was decreased at the high dose, with male and female fetuses equally affected. Live fetal body weight per litter was not affected by administration of DES in corn oil. The incidence of malformations per litter was increased in the high dose group, with females more severely affected than males. This was due to an increase in the incidence of extemal malformations per litter, observed as an increase in the incidence of prominent nipples in the gd 17 fetuses, a characteristic effect of prenatal DES exposure. No significant increase in the incidence of visceral or skeletal malformations or variations was observed for the DES in corn oil-treated litters, except at 5 mg/kg/day DES in corn oil where an increase in skeletal malformations was noted. Malformations included scrambled sternebrae, perforated sternum, and fused ribs, which have not been shown to be specific to DES treatment. Failure to detect a statistically significant number of skeletal malformations in the 10, 50, and 100 mg/kg/day groups may have been influenced by a reduced number of fetuses available for examination in these groups, as a result of prenatal mortality.
For animals dosed with DES in trioctanoin, no maternal lethality was observed. Pregnancy rates were 92% and 100% for the control and 100 mg/kg/day dose group, respectively. Maternal body weight gain was decreased in the 100 mg/kg/day DES in trioctanoin group compared to the trioctanoin control during the gestational period (gd 0-17), and during the treatment period (gd 9-16). Corrected maternal body weight gain was unaffected by treatment with DES in trioctanoin. No treatment-related clinical signs were observed. Maternal relative food consumption was unaffected. Maternal relative water consumption for the DES in trioctanoin was decreased during the period of gd 9-12, but not at any other time interval during treatment. At necropsy, relative maternal liver weight was unaffected, while gravid uterine weight was decreased in the 100 mg/kg/day DES in trioctanoin group. The percent resorptions per litter was increased 5-fold in the 100 mg/kg/day DES in trioctanoin group compared to the trioctanoin control group. Average live litter size was decreased, with male and female fetuses equally affected. Live fetal body weight per litter was decreased by administration of DES in trioctanoin. The incidence of malformations per litter was not increased by DES in trioctanoin treatment. However, when external, visceral, and skeletal malformations were analyzed separately, the incidence of external malformations per litter was increased, due to an increase in the incidence of prominent nipples. No significant increase in the incidence of visceral or skeletal malformations or variations was observed for the DES in trioctanoin-treated litters.
In summary, maternal toxicity was evident as decreased corrected maternal body weight at greater than 2.5 mg/kg/day DES in corn oil. Thus, for DES administered subcutaneously in corn oil to Swiss mice on gd 9-16, a No Observed Adverse Effect Level for maternal toxicity was not determined. Gravid uterine weight and live litter size were decreased at the high dose (100 mg/kg/day). However, the incidence of resorptions (early embryo/fetal death) was increased at greater than 10 mg/kg/day. External malformations were increased at the 100 mg/kg/day, and were characterized by an increase in the incidence of prominent nipples, observed primarily in female fetuses. There was no evidence of altered skeletal development in the fetuses of any dose group. Thus, the lowest observed adverse effect level for developmental toxicity was 10 mg/kg/day and the NOAEL for developmental toxicity was 5 mg/kg/day. Comparison of the effects of 100 mg/kg/day DES administered in corn oil and trioctanoin indicated there was no difference in corn oil or trioctanoin as vehicles for this compound.