Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. Consumption of soy and genistein has been associated with a variety of beneficial effects in animals and humans, but concerns have also been raised concerning potential adverse effects of genistein, particularly with regard to reproductive toxicity and the induction or potentiation of carcinogenesis, due primarily to its weak estrogenic activity. Because of these concerns, genistein was selected as one of the compounds to be examined in a protocol utilizing Sprague-Dawley rats to evaluate the effects of multigenerational and long-term exposures to doses of estrogenic agents that produce subtle reproductive tract lesions in developmentally exposed Sprague-Dawley rat pups. Results from the multigenerational reproductive toxicology feed study are reported in this report, and results of the 2-year feed study are reported separately (NTP, 2008a). Data from a preliminary reproductive dose range-finding feed study (NTP, 2007) that utilized exposure concentrations of up to 1,250 ppm genistein were used to select dietary exposure concentrations of 0, 5, 100, and 500 ppm for the current study. These dietary doses resulted in ingested genistein doses of approximately 0, 0.3, 7, or 35 mg genistein/kg body weight per day for males and 0, 0.5, 10, or 51 mg/kg per day for females during the time that the rats were directly consuming dosed feed. The current study was a multigenerational study (F0 through F4, with F5 litters terminated at weaning) focused on reproductive endpoints. Animals were continuously exposed to genistein 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 sacrificed and necropsied on postnatal day 140 (PND 140). Dosed feed was removed from the F3 pups at the time of weaning, and this generation and subsequent generations were maintained on control feed for the remainder of the study.
For this study, 140 animals of each sex were obtained from the NCTR CD (Sprague-Dawley) rat colony at weaning and placed on a soy- and alfalfa-free diet that was used throughout the study in an attempt to maintain consistently low background exposure to phytoestrogens. Thirty-five animals per sex were assigned to exposure groups by a weight-ranked randomization procedure prior to the start of dietary exposure of the parental (F0) generation at 6 weeks of age. At the time of mating, males were paired with females from the same exposure group, and they were housed together until evidence of successful mating was detected or for a maximum of 14 days. Litters were randomly standardized to four males and four females on PND 2, and 25 litters per exposure group and their associated sires and dams were randomly selected to continue on study to produce the next generation and then necropsied at termination at 20 weeks of age (PND 140). Similar procedures were used to produce each generation.
Results of the current study are summarized below. In the postweaning period, exposure to 500 ppm genistein reduced body weights predominantly in females of generations in which rats were ingesting the compound throughout adulthood (F0 through F2). In the unexposed F4 generation, female body weight was also depressed, although to a lesser extent than in the earlier generations. In the F1 generation, postweaning body weights were reduced in all 100 and 500 ppm groups, with a more pronounced effect in the females. While pup birth weights were not significantly affected by genistein in the F1 through F4 generations (with the exception of 100 ppm males in the F1 generation), both sexes showed depressed body weight gains during the preweaning period in the 500 ppm groups in all of these generations. Male pup preweaning body weight gains were also depressed in the 5 and 100 ppm groups in the F1 generation. In the unexposed F5 generation, pup birth weights in all exposed groups of both sexes were significantly lower than those in the controls, although it seems likely that this is a chance observation rather than a carryover effect from exposures in earlier generations.
Measures of fertility were not adversely affected by genistein except for litter size. Litter size of the 500 ppm group in the F2 generation was significantly smaller than that in the corresponding control group. The litter sizes in the F1, F2, and F3 generations showed negative exposure concentration trends. Male and female 500 ppm pups in the F1 generation had slightly reduced anogenital distances (AGDs) relative to controls when covaried by body weight. Female pups also had reduced AGDs in the F2 (500 ppm) and F3 (100 ppm) generations, although the statistical significance was dependent on the analysis method applied. Females exposed to 500 ppm showed an accelerated time of vaginal opening (approximately 3 days) in the F1 and F2 generations, while the 5 ppm group showed an earlier time of vaginal opening (1.3 days) in the F3 generation. Body weight at vaginal opening was lower in 500 ppm females of the F1 through F3 generations and in the 5 ppm females of the F1 generation. When examined shortly after vaginal opening, estrous cycles of 500 ppm females in the F1 and F2 generations were significantly longer (approximately 3 days and 1 day, respectively) than those of their respective control groups. Other estrous cycle disturbances (with the exception of decreased time in diestrus for 100 ppm females in the F4 generation) were confined to the 500 ppm group of the F1 generation and included reduced time in proestrus and an increase in the number and percentage of aberrant cycles. When the estrous cycles of older animals were examined prior to termination, the sole significant effects were a decreased time in estrus and increased time in diestrus in 5 ppm females of the F2 generation and an increased number of abnormal cycles in 500 ppm females of the F3 generation. No effects of genistein on male sexual development were noted with the exception of an increased time to testicular descent in 500 ppm males of the F3 generation. Significant organ weight effects in both sexes were largely confined to single exposed groups in single generations; no clear patterns indicating toxicity to reproductive or nonreproductive organs were observed.
Exposure-related microscopic lesions were confined to males, with the mammary gland and kidney affected. Incidences of mammary gland alveolar/ductal hyperplasia were significantly increased in 500 ppm males in the F0 through F2 generations and in 100 ppm males in the F1 and F2 generations. In the F3 generation, a significant positive linear exposure concentration trend in the incidences of mammary gland hyperplasia occurred, but no exposed group differed significantly from the controls in pairwise comparisons. The more pronounced effect of genistein on the incidences of male mammary gland hyperplasia in the continuously exposed F1 and F2 generations as compared to the late adolescent and adult exposures of the F0 generation and the preweaning-only exposure of the F3 generation indicates that both developmental and adult exposures contribute to the maintenance of this effect into adulthood. Statistically significant effects of genistein on the incidences of generally minimal to mild kidney lesions in males were confined to the continuously exposed F1 and F2 generations.
Incidences of renal tubule mineralization were significantly increased in 100 and 500 ppm males in the F1 and F2 generations, and incidences of inflammation and renal tubule regeneration were significantly increased in 500 ppm males in the F1 generation.
In addition to the results reported above for animals from the main study, ancillary studies were conducted with pups derived from the current study or from animals treated under similar conditions. These results have been reported elsewhere (Appendix P) and are not presented in detail in this report. Of particular importance are the data on blood and tissue genistein concentrations obtained from adult animals in the F1 generation (Chang et al., 2000), from dams and fetuses (Doerge et al., 2001), and from dams and nursing pups (Doerge et al., 2006). These data provide measures of the internal dose resulting from the dietary exposure concentrations used in the current study and indicate that while fetal and adult exposures to genistein were at concentrations relevant to the full range of human exposures, only very low exposures were achieved during the early neonatal period when the pups were receiving exposures exclusively from the milk. The minimal exposure to genistein during this critical developmental period must be considered in the interpretation of the data derived from the current study.
In summary, although genistein did show adverse effects with dietary exposures of 100 or 500 ppm, there were no clear adverse effects on the reproductive or developmental parameters measured at genistein concentrations ranging from less than 1 ppm (control diet) to 100 ppm, a range of doses producing serum concentrations achievable from the phytoestrogen content of human diets. There were few clear, overtly toxic effects that carried over across directly exposed generations or appeared to be imprinted to carry over into unexposed descendents under the conditions of exposure in this study.
Under the conditions of this study, dietary exposure to 500 ppm genistein (approximately 35 mg genistein/kg body weight per day in males and 51 mg/kg per day in females) decreased body weights, accelerated vaginal opening, decreased anogenital distance, and altered estrous cyclicity in females continuously ingesting genistein. Significant decreases in postweaning body weight and decreases in anogenital distance in males were confined to the F1 generation and were not seen in the similarly exposed F2 generation. In animals exposed to 500 ppm, there was some evidence for reduced litter size in the F1 and F2 generations that were continuously exposed to the test chemical. No other impacts on fertility and no histopathologic lesions were observed in females. The male reproductive tract did not show significant alterations, but increased incidences of hyperplasia of the mammary gland and calcification of renal tubules were observed in continuously exposed 100 and 500 ppm males examined at 20 weeks of age. Weaker effects on the incidences of male mammary gland hyperplasia were observed in 500 ppm males exposed only as adults or exposed only in utero and through lactation. Other than decreased body weight gains in preweaning pups, there was no evidence for a carry over of genistein effects into unexposed generations.