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. A series of short-term studies with genistein was conducted with two goals: 1) to obtain data necessary to establish dose levels for subsequent multigeneration reproductive and chronic toxicity studies and 2) to evaluate the effects of genistein on endpoints outside the reproductive tract. The data generated from these studies have been reported previously in the peer-reviewed literature or in technical reports (Appendix C). In addition, selected data from these studies were analyzed and discussed in the National Toxicology Program's Report of the Endocrine Disruptors Low-Dose Peer Review (NTP, 2001). The present report focuses on the reproductive and general toxicology endpoints evaluated. Data obtained in separate evaluations of behavioral, neuroanatomical, neurochemical, and immunological endpoints, as well as the assessment of serum genistein levels, are also discussed to put in better perspective the selection of doses for the multigenerational and chronic studies.
Genistein was administered in an irradiated soy- and alfalfa-free diet (Purina 5K96) at exposure concentrations of 0, 5, 25, 100, 250, 625, or 1,250 ppm to 10 vaginal plug-positive, female Sprague-Dawley rats starting on gestation day 7 and continuing throughout pregnancy. These dietary exposure concentrations resulted in ingested doses of approximately 0.3, 1.7, 6.4, 16, 38, and 72 mg genistein/kg body weight to dams in the 5, 25, 100, 250, 625, and 1,250 ppm groups, respectively. Dietary exposure of the dams continued through lactation, during which time ingested doses were approximately 0.6, 3.5, 14, 37, 84, and 167 mg/kg per day. Pups from five litters, culled to eight per litter with an equal sex distribution on postnatal day (PND) 2, were maintained on the same dosed feed as their mothers after weaning until sacrifice at PND 50. Ingested doses were approximately 0.6, 3, 11, 29, 69, and 166 mg/kg per day for male pups and 0.6, 3, 12, 31, 73, and 166 mg/kg per day for female pups. Body weight and feed consumption of the treated dams prior to parturition showed decreasing trends with increasing dose, and both parameters were significantly less than those of the controls in the 1,250 ppm group. A significant exposure concentration-related effect on litter birth weight was observed, but no exposed group differed significantly from the control group in pairwise comparisons. Pups in the 1,250 ppm group had significantly decreased body weights relative to controls at the time of sacrifice (males, 9% decrease; females, 12% decrease). The most pronounced organ weight effects in the pups were decreased ventral prostate weight (absolute weight, 28% decrease; relative weight, 20% decrease) in males at 1,250 ppm and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at exposure concentrations greater than 250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at exposure concentrations of 25 ppm or greater for hypertrophy and 250 ppm or greater for hyperplasia. Abnormal cellular maturation (mucocyte metaplasia) in the vagina was observed at 625 and 1,250 ppm, and abnormal ovarian antral follicles were observed at 1,250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1,250 ppm. Histologic evaluation indicated a deficit of sperm in the epididymis at 625 and 1,250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these exposure concentrations. Control females showed a high incidence of renal tubule mineralization, and the severity of this lesion was significantly increased at exposure concentrations of 250 ppm or greater. Males showed no renal tubule mineralization below 250 ppm, but incidence and severity increased with increasing exposure concentration at 250 ppm and greater.
The primary goal of the current study was to provide information for the selection of exposure concentrations to be used in subsequent multigenerational and chronic studies. These long-term studies were designed to address multiple aspects of the endocrine disruptor hypothesis, that is, the hypothesis that exposures of human and wildlife populations to endocrine-active compounds contribute to adverse reproductive tract effects and cancers of hormone-sensitive organs. In particular, the long-term consequences of low dose exposures that may produce subtle initial effects, the magnification of those effects across generations, and the reversibility of those effects were to be investigated. The goal was to select a high exposure concentration that would not induce overt toxicity in the dams or pups but would induce observable effects in the reproductive organs of the pups without severely impairing fertility in the F1 generation. The 1,250 ppm exposure concentration was clearly ruled out for further testing based on the effects on body weights, histopathologic observations in males and females, and a reduction in the proportion of mated dams producing litters. While the effects observed at 625 ppm would not be predicted to significantly impair reproduction, the observation of significant effects at 250 ppm (hyperplasia in the mammary gland of both sexes), together with the suggestion of subtle effects at this exposure concentration and less in the parallel immunotoxicity and neuroanatomical surveys, a high exposure concentration between 250 and 625 ppm was deemed appropriate for the purposes of the multigenerational reproductive toxicology study and the chronic study of genistein. A high exposure concentration for the multigenerational and chronic studies was thus set at 500 ppm. A low exposure concentration of 5 ppm, where no significant effects were observed in the reproductive dose range-finding, and an intermediate exposure concentration of 100 ppm were also selected.