Disposition and metabolism of ethylene glycol 2-ethylhexyl ether in Sprague Dawley rats, B6C3F1/N mice, and in vitro in rat hepatocytes.

Ethylene glycol 2-ethylhexyl ether (EGEHE) is a solvent used in a variety of applications.We report disposition and metabolism of EGEHE following a single gavage or dermal administration of 50, 150 or 500 mg/kg [14C]EGEHE in rats and mice and in vitro in rat hepatocytes.EGEHE was cleared rapidly in rat hepatocytes (half-life ∼4 min) with no sex difference.EGEHE was well- and moderately absorbed following oral administration (rats: 80-96%, mice: 91-95%) and dermal application (rats: 25-37%, mice: 22-24%), respectively, and rapidly excreted in urine.[14C]EGEHE-derived radioactivity was distributed to tissues (oral: 2.3-7.2%, dermal: 0.7-2.2%) with liver and kidney containing the highest levels in both species.EGEHE was extensively metabolised with little to no parent detected in urine. The alkoxyacetic acid metabolite, which has previously been shown to mediate toxicities of other shorter-chain ethylene glycol ethers, was not detected.There were no apparent dose, species or sex differences in disposition and metabolism of EGEHE, except that the exhaled volatile compounds were greater in mice (19-20%) compared with rats (<2%).These studies address a critical gap in the scientific literature and provide data that will inform future studies designed to evaluate toxicity of EGEHE.

Butylparaben multigenerational reproductive assessment by continuous breeding in Hsd:Sprague Dawley SD rats following dietary exposure.

Butylparaben (BP) is an antimicrobial agent utilized for decades as a preservative in numerous consumer products. The safety of parabens has recently come under scrutiny based on reports of estrogenic activity and suggested adverse effects upon the reproductive system. Due to the limited availability of studies that address the potential for BP exposure to induce reproductive toxicity, and clear evidence of human exposure, the National Toxicology Program conducted a multigenerational continuous breeding study to evaluate the impact of dietary BP-exposure at 0, 5000, 15,000, or 40,000 ppm on reproductive and developmental parameters in Hsd:Sprague Dawley SD rats. BP-exposure was not associated with adverse alterations of fertility, fecundity, pubertal attainment, or reproductive parameters in F0, F1, or F2 generations. Exposure-dependent increases in liver weights, and incidences of non-neoplastic liver lesions suggest the liver is a target organ of BP toxicity. No findings were observed that would support the purported mechanism of BP-induced endocrine disruption in perinatally-exposed rodents.

Multigenerational reproductive assessment of 4-methylimidazole administered in the diet to Hsd:Sprague Dawley SD rats.

The general population, including children and adolescents, is exposed to 4-methylimidazole (4-MI) in the diet. 4-MI is a by-product of caramel color manufacturing. It has been previously classified as a possible human carcinogen and displays potential reproductive toxicity. A follow up assessment of reproductive toxicity was conducted in rats utilizing the reproductive assessment by continuous breeding paradigm, in which multiple generations were exposed to 4-MI in diet at 750, 2500, and 5000 ppm. 4-MI exposure was associated with delays in preputial separation and vaginal opening, impairment in reproductive performance, and concomitant histopathological findings in the prostate, testis, and epididymis at 2500 and 5000 ppm. The Lowest Observed Adverse Effect Level for reproductive (based on prostate atrophy) and developmental toxicity (based on delays in preputial separation and vaginal opening) was 750 ppm, equivalent to approximately 50-60 mg/kg bw/day.

Toxicity and carcinogenicity of androstenedione in F344/N rats and B6C3F1 mice.

Androstenedione was marketed as a dietary supplement to increase muscle mass during training. Due to concern over long-term use, the NTP evaluated the subchronic and chronic toxicity and carcinogenicity of androstenedione in male and female F344/N rats and B6C3F1 mice. In subchronic studies, dose limiting effects were not observed. A chronic (2-year) exposure by gavage at 10, 20, or 50 mg/kg in rats and male mice, and 2, 10, or 50 mg/kg in female mice (50 mg/kg, maximum feasible dose) was conducted. Increased incidences of lung alveolar/bronchiolar adenoma and carcinoma occurred in the 20 mg/kg male rats and increases in mononuclear cell leukemia occurred in the 20 and 50 mg/kg female rats, which may have been related to androstenedione administration. In male and female mice, androstenedione was carcinogenic based upon a significant increase in hepatocellular tumors. A marginal increase in pancreatic islet cell adenomas in male (50 mg/kg) and female (2, 10, 50 mg/kg) mice was considered to be related to androstenedione administration. Interestingly, incidences of male rat Leydig cell adenomas and female rat mammary gland fibroadenomas decreased. In conclusion, androstenedione was determined to be carcinogenic in male and female mice, and may have been carcinogenic in rats.

Fifteen years after "Wingspread"--environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go.

In 1991, a group of expert scientists at a Wingspread work session on endocrine-disrupting chemicals (EDCs) concluded that "Many compounds introduced into the environment by human activity are capable of disrupting the endocrine system of animals, including fish, wildlife, and humans. Endocrine disruption can be profound because of the crucial role hormones play in controlling development." Since that time, there have been numerous documented examples of adverse effects of EDCs in invertebrates, fish, wildlife, domestic animals, and humans. Hormonal systems can be disrupted by numerous different anthropogenic chemicals including antiandrogens, androgens, estrogens, AhR agonists, inhibitors of steroid hormone synthesis, antithyroid substances, and retinoid agonists. In addition, pathways and targets for endocrine disruption extend beyond the traditional estrogen/androgen/thyroid receptor-mediated reproductive and developmental systems. For example, scientists have expressed concern about the potential role of EDCs in increasing trends in early puberty in girls, obesity and type II diabetes in the United States and other populations. New concerns include complex endocrine alterations induced by mixtures of chemicals, an issue broadened due to the growing awareness that EDCs present in the environment include a variety of potent human and veterinary pharmaceutical products, personal care products, nutraceuticals and phytosterols. In this review we (1) address what have we learned about the effects of EDCs on fish, wildlife, and human health, (2) discuss representative animal studies on (anti)androgens, estrogens and 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals, and (3) evaluate regulatory proposals being considered for screening and testing these chemicals.

Iprodione delays male rat pubertal development, reduces serum testosterone levels, and decreases ex vivo testicular testosterone production.

Iprodione (IPRO) is a dichlorophenyl dicarboximide fungicide similar to procymidone and vinclozolin. All three of these fungicides induce Leydig cell tumors in the rat testis in long-term studies and an endocrine mode of action has been hypothesized to mediate this effect. Although both procymidone and vinclozolin antagonize the androgen receptor (AR) in vitro and in vivo, IPRO does not appear to be an AR antagonist. We proposed that pubertal exposure to IPRO would delay male rat pubertal development and reduce testosterone production within the testis. Sprague-Dawley weanling rats were dosed by gavage with 0, 50, 100, or 200mg/kg/day of IPRO from post-natal day (PND) 23 to 51/52. The onset of puberty (progression of preputial separation (PPS)) was measured starting on PND 37. Organ weights, serum hormones, and ex vivo testis steroid hormone production under stimulated (+human chorionic gonadotropin (hCG)) and unstimulated (-hCG) conditions were measured at necropsy. IPRO delayed PPS at 100 and 200mg/kg/day and decreased androgen sensitive seminal vesicle and epididymides weights at 200mg/kg/day. Furthermore, IPRO increased adrenal and liver weights at 200mg/kg/day, presumably by different mechanism(s) of action. Serum testosterone levels were decreased along with serum 17alpha-hydroxyprogesterone and androstenedione whereas serum LH was unaffected. IPRO reduced ex vivo testis production of testosterone and progesterone. Taken together, these results suggest that IPRO affects steroidogenesis within the testis, not through disruption of LH signaling, but possibly through enzyme inhibition of the steroidogenic pathway before CYP17. These data, along with the reported failure of IPRO to elicit an AR antagonism in vitro, provide evidence that IPRO differs from the dicarboximides procymidone and vinclozolin in that the effects on male rat pubertal development result from an inhibition of steroidogenesis and not AR antagonism.

Sensitivity of fetal rat testicular steroidogenesis to maternal prochloraz exposure and the underlying mechanism of inhibition.

The fungicide prochloraz (PCZ) induces malformations in androgen-dependent tissues in male rats when administered during sex differentiation. The sensitivity of fetal testicular steroidogenesis to PCZ was investigated to test the hypothesis that the reported morphological effects from maternal exposure were associated with reduced testosterone synthesis. Pregnant Sprague-Dawley rats were dosed by gavage with 0, 7.8, 15.6, 31.3, 62.5, and 125 mg PCZ/kg/day (n = 8) from gestational day (GD) 14 to 18. On GD 18, the effects of PCZ on fetal steroidogenesis were assessed by measuring hormone production from ex vivo fetal testes after a 3-h incubation. Lastly, PCZ levels in amniotic fluid and maternal serum were measured using liquid chromatography/mass spectroscopy and correlated to the inhibition of steroidogenesis. Fetal progesterone and 17alpha-hydroxyprogesterone production levels were increased significantly at every PCZ dose, whereas testosterone levels were significantly decreased only at the two high doses. These results suggest that PCZ inhibits the conversion of progesterone to testosterone through the inhibition of CYP17. To test this hypothesis, PCZ effects on CYP17 gene expression and in vitro CYP17 hydroxylase activity were evaluated. PCZ had no effect on testicular CYP17 mRNA levels as measured by quantitative real-time polymersase chain reaction. However, microsomal CYP17 hydroxylase activity was significantly inhibited by the fungicide (K(i) = 865nM). Amniotic fluid PCZ concentrations ranged from 78 to 1512 ppb (207-4014nM) and testosterone production was reduced when PCZ reached approximately 500 ppb, which compares favorably with the determined CYP17 hydroxylase K(i) (326 ppb). These results demonstrate that PCZ lowers testicular testosterone synthesis by inhibiting CYP17 activity which likely contributes to the induced malformations in androgen-dependent tissues of male offspring.

Prochloraz inhibits testosterone production at dosages below those that affect androgen-dependent organ weights or the onset of puberty in the male Sprague Dawley rat.

Prochloraz (PCZ) is an imidazole fungicide that inhibits gonadal steroidogenesis and antagonizes the androgen receptor (AR). We hypothesized that pubertal exposure to PCZ would reduce testosterone production and delay male rat reproductive development. Sprague Dawley rats were dosed by gavage with 0, 31.3, 62.5, or 125 mg/kg/day of PCZ from postnatal day (PND) 23 to 42 or 51. There was a significant delay in preputial separation (PPS) at 125 mg/kg/day PCZ and several of the androgen-dependent organ weights were decreased significantly, but the significant organ weight effects were not consistent between the 2 necropsies (PND 42 vs. 51). At both ages, serum testosterone levels and ex vivo testosterone release from the testis were significantly decreased whereas serum progesterone and 17alpha-hydroxyprogesterone levels were significantly increased at dose levels below those that affected PPS or reproductive organ weights. The hormone results suggested that PCZ was inhibiting CYP17 activity. In a second pubertal study (0, 3.9, 7.8, 15.6, 31.3, or 62.5 mg/kg/day PCZ), serum testosterone levels and ex vivo testosterone production were significantly reduced at 15.6 mg/kg/day PCZ. In order to examine the AR antagonism effects of PCZ, independent of its effects on testosterone synthesis, castrated immature male rats were dosed with androgen and 0, 15.6, 31.3, 62.5, or 125 mg/kg/day PCZ for 10-11 days (Hershberger assay). In this assay, androgen-sensitive organ weights were only significantly decreased at 125 mg/kg/day PCZ. These data from the pubertal assays demonstrate that PCZ decreases testosterone levels and delays rat pubertal development, as hypothesized. However, the fact that hormone levels were affected at dosage eightfold below that which delayed the onset of puberty suggests that rather large reductions in serum testosterone may be required to delay puberty and consistently reduce androgen-dependent tissue weights.

Effect of conazole fungicides on reproductive development in the female rat.

Three triazole fungicides were evaluated for effects on female rat reproductive development. Rats were exposed via feed to propiconazole (P) (100, 500, or 2500 ppm), myclobutanil (M) (100, 500, or 2000 ppm), or triadimefon (T) (100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 98. Body weight (BW) and anogenital distance (AGD) at PND 0, age and BW at vaginal opening (VO), estrous cyclicity, and body and organ weight at necropsy were measured. BW at PND 0 was unaffected by treatment. AGD was increased by M2000. VO was delayed by M2000 and T1800. Estrous cyclicity was initially disrupted by P500, P2500 and T1800, but later normalized. At PND 99 there was a decrease in BW by T1800, an increase in liver weight by P2500 and T1800, and an increase in ovarian weight by M2000 and T1800. It is concluded that exposure to P, M and T adversely impacted female rodent reproductive development.