EDC IMPACT: Reduced sperm counts in rats exposed to human relevant mixtures of endocrine disrupters.

Human semen quality is declining in many parts of the world, but the causes are ill defined. In rodents, impaired sperm production can be seen with early life exposure to certain endocrine-disrupting chemicals, but the effects of combined exposures are not properly investigated. In this study, we examined the effects of early exposure to the painkiller paracetamol and mixtures of human relevant endocrine-disrupting chemicals in rats. One mixture contained four estrogenic compounds; another contained eight anti-androgenic environmental chemicals and a third mixture contained estrogens, anti-androgens and paracetamol. All exposures were administered by oral gavage to time-mated Wistar dams rats (n = 16-20) throughout gestation and lactation. In the postnatal period, testicular histology was affected by the total mixture, and at the end of weaning, male testis weights were significantly increased by paracetamol and the high doses of the total and the anti-androgenic mixture, compared to controls. In all dose groups, epididymal sperm counts were reduced several months after end of exposure, i.e. at 10 months of age. Interestingly, the same pattern of effects was seen for paracetamol as for mixtures with diverse modes of action. Reduced sperm count was seen at a dose level reflecting human therapeutic exposure to paracetamol. Environmental chemical mixtures affected sperm count at the lowest mixture dose indicating an insufficient margin of safety for the most exposed humans. This causes concern for exposure of pregnant women to paracetamol as well as environmental endocrine disrupters.

Low-dose effect of developmental bisphenol A exposure on sperm count and behaviour in rats.

Bisphenol A is widely used in food contact materials and other products and is detected in human urine and blood. Bisphenol A may affect reproductive and neurological development; however, opinion of the European Food Safety Authority (EFSA) on bisphenol A (EFSA J, 13, 2015 and 3978) concluded that none of the available studies were robust enough to provide a point of departure for setting a tolerable daily intake for bisphenol A. In the present study, pregnant Wistar rats (n = 17-21) were gavaged from gestation day 7 to pup day 22 with bisphenol A doses of 0, 25 µg, 250 µg, 5 mg or 50 mg/kg bw/day. In the offspring, growth, sexual maturation, weights and histopathology of reproductive organs, oestrus cyclicity and sperm counts were assessed. Neurobehavioural development was investigated using a behavioural testing battery including tests for motor activity, sweet preference, anxiety and spatial learning. Decreased sperm count was found at the lowest bisphenol A dose, that is 25 µg/kg/day, but not at the higher doses. Reproductive organ weight and histology were not affected and no behavioural effects were seen in male offspring. In the female offspring, exposure to 25 µg/kg bw/day bisphenol A dose resulted in increased body weight late in life and altered spatial learning in a Morris water maze, indicating masculinization of the brain. Decreased intake of sweetened water was seen in females from the highest bisphenol A dose group, also a possible sign of masculinization. The other investigated endpoints were not significantly affected. In conclusion, the present study using a robust experimental study design, has shown that developmental exposure to 25 µg/kg bw/day bisphenol A can cause adverse effects on fertility (decreased sperm count), neurodevelopment (masculinization of spatial learning in females) and lead to increased female body weight late in life. These results suggest that the new EFSA temporary tolerable daily intake of 4 µg/kg bw/day is not sufficiently protective with regard to endocrine disrupting effects of bisphenol A in humans.

Multiple Endocrine Disrupting Effects in Rats Perinatally Exposed to Butylparaben.

Parabens comprise a group of preservatives commonly added to cosmetics, lotions, and other consumer products. Butylparaben has estrogenic and antiandrogenic properties and is known to reduce sperm counts in rats following perinatal exposure. Whether butylparaben exposure can affect other endocrine sensitive endpoints, however, remains largely unknown. In this study, time-mated Wistar rats (n = 18) were orally exposed to 0, 10, 100, or 500 mg/kg bw/d of butylparaben from gestation day 7 to pup day 22. Several endocrine-sensitive endpoints were adversely affected. In the 2 highest dose groups, the anogenital distance of newborn male and female offspring was significantly reduced, and in prepubertal females, ovary weights were reduced and mammary gland outgrowth was increased. In male offspring, sperm count was significantly reduced at all doses from 10 mg/kg bw/d. Testicular CYP19a1 (aromatase) expression was reduced in prepubertal, but not adult animals exposed to butylparaben. In adult testes, Nr5a1 expression was reduced at all doses, indicating persistent disruption of steroidogenesis. Prostate histology was altered at prepuberty and adult prostate weights were reduced in the high dose group. Thus, butylparaben exerted endocrine disrupting effects on both male and female offspring. The observed adverse developmental effect on sperm count at the lowest dose is highly relevant to risk assessment, as this is the lowest observed adverse effect level in a study on perinatal exposure to butylparaben.

Mixtures of endocrine disrupting contaminants modelled on human high end exposures: an exploratory study in rats.

By diminishing the action of androgens during gestation, certain chemicals can induce irreversible demasculinization and malformations of sex organs in the male rat after gestational exposure. Studies with mixtures of such anti-androgens have shown that substantial combined effects occur even though each individual chemical is present at low, ineffective doses, but the effects of mixtures modelled based on human intakes have not previously been investigated. To address this issue for the first time, we selected 13 chemicals for a developmental mixture toxicity study in rats where data about in vivo endocrine disrupting effects and information about human exposures was available, including phthalates, pesticides, UV-filters, bisphenol A, parabens and the drug paracetamol. The mixture ratio was chosen to reflect high end human intakes. To make decisions about the dose levels for studies in the rat, we employed the point of departure index (PODI) approach, which sums up ratios between estimated exposure levels and no-observed-adverse-effect-level (NOAEL) values of individual substances. For high end human exposures to the 13 selected chemicals, we calculated a PODI of 0.016. As only a PODI exceeding 1 is expected to lead to effects in the rat, a total dose more than 62 times higher than human exposures should lead to responses. Considering the high uncertainty of this estimate, experience on lowest-observed-adverse-effect-level (LOAEL)/NOAEL ratios and statistical power of rat studies, we expected that combined doses 150 times higher than high end human intake estimates should give no, or only borderline effects, whereas doses 450 times higher should produce significant responses. Experiments indeed showed clear developmental toxicity of the 450-fold dose in terms of increased nipple retention (NR) and reduced ventral prostate weight. The 150-fold dose group exhibited significantly increased NR. These observations suggest that highly exposed population groups, especially women of reproductive age, may not be protected sufficiently against the combined effects of chemicals that affect the hormonal milieu required for normal male sexual differentiation.

Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat.

The incidence of hypospadias is increasing in young boys, but it remains unclear whether human exposure to endocrine disrupting chemicals plays a role. Risk assessment is based on estimation of no-observed-adverse-effect levels for single compounds, although humans are exposed to combinations of several anti-androgenic chemicals. In a mixture (MIX) study with three androgen receptor antagonists, vinclozolin, flutamide and procymidone, rats were gavaged during gestation and lactation with several doses of a MIX of the three chemicals or the chemicals alone. External malformations of the male reproductive organs were assessed on PND 47 using a score from 0 to 3 (normal to marked) for hypospadias. Markedly increased frequencies were observed after exposure to a MIX of the three chemicals compared to administration of the three chemicals alone. Anogenital distance at PND 1, nipple retention at PND 13, and dysgenesis score at PND 16 were highly correlated with the occurrence of hypospadias, and MIX effects were seen at doses where each of the individual chemicals caused no observable effects. Therefore, the results indicate that doses of anti-androgens, which appear to induce no hypospadias when judged on their own, may induce a very high frequency of hypospadias when they interact in concert with other anti-androgens.

Endocrine-disrupting properties in vivo of widely used azole fungicides.

The endocrine-disrupting potential of four commonly used azole fungicides, propiconazole, tebuconazole, epoxiconazole and ketoconazole, were tested in two short-term in vivo studies. Initially, the antiandrogenic effects of propiconazole and tebuconazole (50, 100 and 150 mg/kg body weight/day each) were examined in the Hershberger assay. In the second study, pregnant Wistar rats were dosed with propiconazole, tebuconazole, epoxiconazole or ketoconazole (50 mg/kg/day each) from gestational day (GD) 7 to GD 21. Caesarian sections were performed on dams at GD 21. Tebuconazole and propiconazole demonstrated no antiandrogenic effects at doses between 50 and 150 mg/kg body weight/day in the Hershberger assay. In the in utero exposure toxicity study, ketoconazole, a pharmaceutical to treat human fungal infections, decreased anogenital distance and reduced testicular testosterone levels, demonstrating a demasculinizing effect on male fetuses. Tebuconazole, epoxiconazole and ketoconazole induced a high-frequency of post-implantation loss, and both ketoconazole and epoxiconazole caused a marked increase in late and very late resorptions. Overall the results show that many of the commonly used azole fungicides act as endocrine disruptors in vivo, although the profile of action in vivo varies. As ketoconazole is known to implicate numerous endocrine-disrupting effects in humans, the concern for the effects of the other tested azole fungicides in humans is growing.