Reproductive safety studies with genistein in rats.

Genistein is a phytoestrogen that occurs naturally in the diet and is found in a wide variety of plant-derived foods especially in soybeans and soy-based foods. There is wide spread interest in genistein and related phytoestrogens as chemopreventive agents for a variety of human diseases and cancers based on epidemiologic evidence of reduced cancer rates in populations with a high intake of soy. Soy, and hence its constituents, such as genistein, have been consumed at high levels in several Asian populations for many centuries without any apparent adverse effects and to the contrary, many health benefits have been associated with the ingestion of soy based foods. Concern has been raised, however, of potential adverse effects due to the estrogenic and other activities of the isoflavones and thus a comprehensive series of safety studies was performed with genistein. To assess the teratogenic and fetal toxic potential of genistein, several studies were conducted. Genistein was tested in an in vitro rat whole embryo culture assay (WEC), which is a preliminary screen, for fetotoxic and teratogenic potential, over a concentration range of from 1 to 100 microg/mL. Treatment related anomalies were observed at concentrations of >or= 10 microg and at 100 microg/mL, all embryos were malformed. Two in vivo embryo fetal developmental safety studies were conducted with genistein by oral administration (gavage and dietary admix) in which there was no evidence for a teratogenic effect. In an oral (gavage) embryonic and fetal development pilot study, genistein was administered to rats at dose levels of 0, 20, 150 and 1000 mg/kg/day from days 6-20 of gestation to females that were allowed to litter and rear their offspring up to day 7 of lactation. A slight maternal toxicity at 1000 mg/kg/day was observed as indicated by decreased body weight and food consumption and at this dose, adverse effects in the pups were observed including increased pup mortality, poor general condition, reduced pup body weights, and reduced pup milk uptake. At the high dose of 1000 mg/kg, no external malformations were noted, however some minor visceral and skeletal variations were observed. At the low dose of 20 mg/kg/day, an increased mortality, reduced milk uptake, a decreased % male sex ratio, and decreased body weights during lactation were observed. Due to lack of effects at the mid dose and the small number of animals, a relationship to treatment was considered unlikely. In an oral (dietary admix) Prenatal developmental safety study, genistein was administered to rats at dose levels of 0, 5, 50, 100 and 500 mg/kg/day from day 5-21 of gestation. At 500 mg/kg, maternal body weight and food consumption were markedly reduced. The incidence of resorptions was markedly increased with a corresponding decrease in the number of live fetuses per dam. Fetal body weights were also reduced. No treatment-related teratogenic effects were noted during external, visceral and skeletal examination of fetuses or in body weight normalized anogenital distance. On the basis of these studies, it is concluded that genistein has no teratogenic potential in vivo at very high doses of up to 1000 mg/kg/day by oral gavage in the embryo-fetal toxicity pilot study or up to 500 mg/kg/day by dietary admix in the Prenatal developmental study even though these doses were maternally toxic and fetal-toxic. In vitro, genistein had teratogenic potential at high concentrations in the WEC screening assay, however this was not predictive of the in vivo findings. On the basis of the definitive Prenatal development study, the NOAEL for maternal toxicity and adverse effects on embryonic development was considered to be 100 mg/kg/day when administered orally by dietary admix.

Genetic toxicity studies with genistein.

Genistein is a phytoestrogen that occurs naturally in the diet especially in soybeans and soy-based foods. Genistein and related phytoestrogens are of interest as chemopreventive agents for a variety of diseases and cancers based on epidemiologic evidence of reduced cancer rates in populations with a high intake of soy. Although soy and its constituents have been consumed at high levels in Asian populations without apparent adverse effects, concern has been raised of potential adverse effects due to estrogenic and other activities of the isoflavones. In these studies, genistein was evaluated for mutagenicity and clastogenicity in vitro in the S. typhimurium assay (Ames Test), the mouse lymphoma assay and in vivo in the micronucleus test in mice and rats. There was no evidence for a mutagenic effect in the in vitro S. typhimurium assay with and without metabolic activation (S9). In the in vitro mouse lymphoma assay, genistein increased resistant mutants with and without metabolic activation (S9), which were predominantly small colonies indicating that genistein acts as a clastogen. Three independent in vivo micronucleus tests were performed in Moro mice, RAIf rats and Wistar rats. MORO male and female mice were treated orally for 14 days at doses up to 20 mg/kg/day. RAIf and Wistar male and female rats were treated orally at doses up to 2000 mg/kg without an increase in micronuclei in treated mice or rats. It is concluded that genistein was not mutagenic in the S. typhimurium assay or mutagenic or clastogenic in vivo in the mouse and rat micronucleus test. In the mouse lymphoma assay, genistein induced an increase of predominantly small colonies indicating that genistein acts as a clastogen. This observation is in agreement with published data on the inhibitory action of genistein on topoisomerase II, which is known to lead to chromosomal damage with a threshold dose response.

Acute, subchronic and chronic safety studies with genistein in rats.

Genistein is a phytoestrogen that occurs naturally in the diet, especially in soy based foods. There is wide spread interest in phytoestrogens as chemopreventive agents for a variety of diseases and cancers based on epidemiologic evidence. Although soy, and its constituents such as genistein, have been consumed at high levels in several Asian populations without apparent adverse effects, concern has been raised about potential adverse effects due to the estrogenic and other activities. Safety studies with genistein were conducted in the Wistar rat including two acute studies, two subchronic (4 weeks and 13 weeks) and a chronic 52-week dietary admix study. In the acute studies, genistein had a low order of toxicity. In the three repeated dose safety studies at doses up to 500 mg/kg/day, genistein was well tolerated. In all of the studies, decreased food consumption and body weight gain were observed at 500 mg/kg/day. The main hematological findings were decreased red blood cell parameters at 500 mg/kg/day with a compensatory increase in reticulocytes. For clinical chemistry, with the exception of a slight increase in gamma glutamyl transferase in male and female rats at the high dose, there were a number of other minor changes considered not toxicologically significant. At necropsy, there were relatively few macroscopic changes; in the 52-week study, dilation of the uterus with fluid at the high dose and cysts of the ovaries in treated animals were observed. Organ weight changes in male rats at the high dose of 500 mg/kg/day included increased kidney, spleen, adrenal and testes weights and for females included, increased liver, kidney, spleen, ovary and uterus weights. After 4 and 13 weeks of treatment with genistein, there were no treatment related histopathologic findings. After 26 and 52 weeks of treatment, histological changes were seen in the female reproductive organs (ovaries and uterus), and in males (epididymides and prostate), and bone, kidneys, heart, liver and spleen in both sexes. After 52 weeks of treatment of males, vacuolation of the epididymal epithelium at 500 mg/kg/day and inflammation of the prostate were recorded at a higher incidence at 50 and 500 mg/kg/day. In females, cytological changes in the uterus, squamous metaplasia at 50 and 500 mg/kg/day and hyperplasia at 500 mg/kg/day were observed. Furthermore, hydrometra of the uterus and findings in the vagina consisting of anestric or diestrus vaginal mucosa with vaginal mucification, hyperplastic epithelium and multifocal cystic degeneration were noted at 500 mg/kg/day. Atrophy of the ovaries increased in severity in animals at 50 and 500 mg/kg/day. Osteopetrosis (hyperostosis) was observed in male and female rats at 50 and 500 mg/kg/day along with a compensatory increase in extramedullary hemopoiesis in the spleen; females were more affected than males. Hepatocellular hypertrophy and minimal bile duct proliferation were recorded at a higher incidence in animals at 500 mg/kg/day. It is concluded that almost all of the treatment related findings in these studies are related to the estrogenic properties of genistein as a phytoestrogen and would be expected to occur with a compound with estrogenic activity. The hormonally related changes were considered to be functional in nature and thus not adverse effects. Most of the findings in these studies were limited to the high dose of 500 mg/kg/day and were reversible. The few findings observed at 50 mg/kg/day were relatively minor and in view of the functional (hormonally mediated) nature of the effects, were considered not adverse effects. The increased incidence of minimal bile duct proliferation and slightly increased gamma glutamyl transferase are indicative of a mild hepatic effect at the high dose of 500 mg/kg/day. The no observed adverse effect level (NOAEL) of genistein is considered to be 50 mg/kg/day based on the presence of mild hepatic effects at the high dose of 500 mg/kg/day. The no observed effect level (NOEL) is considered to be 5 mg/kg/day based on the hormonally induced functional changes at higher doses.

Subchronic and chronic safety studies with genistein in dogs.

Genistein is a phytoestrogen that occurs naturally in the diet, especially in soy-based foods. There is widespread interest in phytoestrogens as chemopreventive agents for a variety of diseases and cancers based on epidemiologic evidence. Although soy and its constituents, such as genistein, have been consumed at high levels in several Asian populations without apparent adverse effects, concern has been raised about potential adverse effects due to estrogenic and other activities. The subchronic and chronic safety of genistein were evaluated in the beagle dog including a 4-week study and a 52-week safety study with a 13 week interim sacrifice and a 4 week recovery period. In both studies at doses of 50, 150 and 500 mg/kg/day, genistein was well tolerated. In the 4 week study, except for an increase in uterine weights in female dogs at 500 mg/kg/day, there were no other treatment related findings. In the 52-week study, the primary effects of genistein were observed on the reproductive tract, which included for male dogs: reduced size and/or weight of the testes, epididymus and prostate of 2/2 dogs after 13 weeks of treatment and in 1/4 dogs after 52 weeks of treatment at 500 mg/kg/day. The histological changes observed in the affected dogs at 500 mg/kg/day indicated atrophy of the testes and prostate gland and absent spermatozoa in the epididymus. At the mid-dose of 150 mg/kg/day, although there was a reduction to a lesser extent in testes weight after 13, but not 52 weeks, there were no histopathological changes. In female dogs, the reproductive tract effects included increased uterine weight at 500 mg/kg/day after 13 weeks of treatment, but not after 52 weeks of treatment. There was also a small decrease in ovarian weights at 150 and 500 mg/kg/day after 13 weeks and at 500 mg/kg/day after 52 weeks of treatment. There were no histopathological correlates to the changes in organ weights in female dogs. In the 4-week recovery group dogs, no changes were observed in dogs previously treated for 52 weeks with 500 mg/kg/day of genistein. It is concluded that the administration of genistein to dogs for a period of 4-52 weeks was well tolerated and did not result in systemic toxicity. Effects of genistein on the reproductive tract at very high doses were functional in nature and are of a type that would be expected in view of the relatively weak estrogenic activity of genistein and were considered not adverse effects. In the 4-week study, the no observed adverse effect level (NOAEL) for genistein was considered to be >500 mg/kg/day and the no observed effect level (NOEL) was considered to be 150 mg/kg/day. For the 52-week study, the NOAEL is considered to be >500 mg/kg/day and the NOEL is considered to be 50 mg/kg/day.