Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders.

Male reproductive development is intricately dependent on fetal androgen action. Consequently, disrupted androgen action during fetal life can interfere with the development of the reproductive system resulting in adverse effects on reproductive function later in life. One biomarker used to evaluate fetal androgen action is the anogenital distance (AGD), the distance between the anus and the external genitalia. A short male AGD is strongly associated with genital malformations at birth and reproductive disorders in adulthood. AGD is therefore used as an effect readout in rodent toxicity studies aimed at testing compounds for endocrine activity and anti-androgenic properties, and in human epidemiological studies to correlate fetal exposure to endocrine disrupting chemicals to feminization of new-born boys. In this review, we have synthesized current data related to intrauterine exposure to xenobiotics and AGD measurements. We discuss the utility of AGD as a retrospective marker of in utero anti-androgenicity and as a predictive marker for male reproductive disorders, both with respect to human health and rodent toxicity studies. Finally, we highlight four areas that need addressing to fully evaluate AGD as a biomarker in both a regulatory and clinical setting.

Scientific principles for the identification of endocrine-disrupting chemicals: a consensus statement.

Endocrine disruption is a specific form of toxicity, where natural and/or anthropogenic chemicals, known as "endocrine disruptors" (EDs), trigger adverse health effects by disrupting the endogenous hormone system. There is need to harmonize guidance on the regulation of EDs, but this has been hampered by what appeared as a lack of consensus among scientists. This publication provides summary information about a consensus reached by a group of world-leading scientists that can serve as the basis for the development of ED criteria in relevant EU legislation. Twenty-three international scientists from different disciplines discussed principles and open questions on ED identification as outlined in a draft consensus paper at an expert meeting hosted by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany on 11-12 April 2016. Participants reached a consensus regarding scientific principles for the identification of EDs. The paper discusses the consensus reached on background, definition of an ED and related concepts, sources of uncertainty, scientific principles important for ED identification, and research needs. It highlights the difficulty in retrospectively reconstructing ED exposure, insufficient range of validated test systems for EDs, and some issues impacting on the evaluation of the risk from EDs, such as non-monotonic dose-response and thresholds, modes of action, and exposure assessment. This report provides the consensus statement on EDs agreed among all participating scientists. The meeting facilitated a productive debate and reduced a number of differences in views. It is expected that the consensus reached will serve as an important basis for the development of regulatory ED criteria.

A proposed framework for the systematic review and integrated assessment (SYRINA) of endocrine disrupting chemicals.

BACKGROUND: The issue of endocrine disrupting chemicals (EDCs) is receiving wide attention from both the scientific and regulatory communities. Recent analyses of the EDC literature have been criticized for failing to use transparent and objective approaches to draw conclusions about the strength of evidence linking EDC exposures to adverse health or environmental outcomes. Systematic review methodologies are ideal for addressing this issue as they provide transparent and consistent approaches to study selection and evaluation. Objective methods are needed for integrating the multiple streams of evidence (epidemiology, wildlife, laboratory animal, in vitro, and in silico data) that are relevant in assessing EDCs. METHODS: We have developed a framework for the systematic review and integrated assessment (SYRINA) of EDC studies. The framework was designed for use with the International Program on Chemical Safety (IPCS) and World Health Organization (WHO) definition of an EDC, which requires appraisal of evidence regarding 1) association between exposure and an adverse effect, 2) association between exposure and endocrine disrupting activity, and 3) a plausible link between the adverse effect and the endocrine disrupting activity. RESULTS: Building from existing methodologies for evaluating and synthesizing evidence, the SYRINA framework includes seven steps: 1) Formulate the problem; 2) Develop the review protocol; 3) Identify relevant evidence; 4) Evaluate evidence from individual studies; 5) Summarize and evaluate each stream of evidence; 6) Integrate evidence across all streams; 7) Draw conclusions, make recommendations, and evaluate uncertainties. The proposed method is tailored to the IPCS/WHO definition of an EDC but offers flexibility for use in the context of other definitions of EDCs. CONCLUSIONS: When using the SYRINA framework, the overall objective is to provide the evidence base needed to support decision making, including any action to avoid/minimise potential adverse effects of exposures. This framework allows for the evaluation and synthesis of evidence from multiple evidence streams. Finally, a decision regarding regulatory action is not only dependent on the strength of evidence, but also the consequences of action/inaction, e.g. limited or weak evidence may be sufficient to justify action if consequences are serious or irreversible.

Perfluorononanoic acid in combination with 14 chemicals exerts low-dose mixture effects in rats.

Humans are simultaneously exposed to several chemicals that act jointly to induce mixture effects. At doses close to or higher than no-observed adverse effect levels, chemicals usually act additively in experimental studies. However, we are lacking knowledge on the importance of exposure to complex real-world mixtures at more relevant human exposure levels. We hypothesised that adverse mixture effects occur at doses approaching high-end human exposure levels. A mixture (Mix) of 14 chemicals at a combined dose of 2.5 mg/kg bw/day was tested in combination with perfluorononanoic acid (PFNA) at doses of 0.0125 (Low PFNA), 0.25 (Mid PFNA) and 5 (High PFNA) mg/kg bw/day by oral administration for 14 days in juvenile male rats. Indication of a toxicokinetic interaction was found, as simultaneous exposure to PFNA and the Mix caused a 2.8-fold increase in plasma PFNA concentrations at Low PFNA. An increase in testosterone and dihydrotestosterone plasma concentrations was observed for Low PFNA + Mix. This effect was considered non-monotonic, as higher doses did not cause this effect. Reduced LH plasma concentrations together with increased androgen concentrations indicate a disturbed pituitary-testis axis caused by the 15-chemical mixture. Low PFNA by itself increased the corticosterone plasma concentration, an effect which was normalised after simultaneous exposure to Mix. This combined with affected ACTH plasma concentrations and down-regulation of 11ß HSD mRNA in livers indicates a disturbed pituitary-adrenal axis. In conclusion, our data suggest that mixtures of environmental chemicals at doses approaching high-end human exposure levels can cause a hormonal imbalance and disturb steroid hormones and their regulation. These effects may be non-monotonic and were observed at low doses. Whether this reflects a more general phenomenon that should be taken into consideration when predicting human mixture effects or represents a rarer phenomenon remains to be shown.

Perinatal exposure to mixtures of anti-androgenic chemicals causes proliferative lesions in rat prostate.

BACKGROUND: Elevated levels of endogenous or exogenous estrogens during fetal life can induce permanent disturbances in prostate growth and predispose to precancerous lesions. Recent studies have indicated that also early anti-androgen exposure may affect prostate cancer risk. METHODS: We examined the influence of perinatal exposure to mixtures of anti-androgenic and estrogenic chemicals on prostate development. Wistar rats were exposed from gestation day 7 to postnatal day 22 to a mixture of 8 anti-androgenic compounds (AAMix), a mixture of four estrogenic compounds (EMix), or paracetamol or a mixture of all 13 compounds (TotalMix) in mixture ratios reflecting human exposure levels. RESULTS: Ventral prostate weights were reduced by the TotalMix and AAMix in pre-pubertal rats. Histological changes in prostate appeared with increasing age and indicated a shift from the normal age-dependent epithelial atrophy towards hyperplasia. These lesions showed similarities to pre-cancerous lesions in humans. Increased proliferation was observed already in pre-puberty and it was hypothesized that this could be associated with reduced ERß signaling, but no clear conclusions could be made from gene expression studies on ERß-related pathways. The influences of the estrogenic chemicals and paracetamol on prostate morphology were minor, but in young adulthood the estrogen mixture reduced ventral prostate mRNA levels of Igf1 and paracetamol reduced the mRNA level ofPbpc3. CONCLUSIONS: Mixtures of endocrine disrupters relevant for human exposure was found to elicit persistent effects on the rat prostate following perinatal exposure, suggesting that human perinatal exposure to environmental chemicals may increase the risk of prostate cancer later in life.

Low-dose effects of bisphenol A on early sexual development in male and female rats.

Bisphenol A (BPA) is widely detected in human urine and blood. BPA has been reported to impair many endpoints for reproductive and neurological development; however, it is controversial whether BPA has effects in the microgram per kilogram dose range. The aim of the current study was to examine the influence of BPA on early sexual development in male and female rats at dose levels covering both regulatory no observed adverse effect levels (NOAELs) (5 and 50 mg/kg bw per day) as well as doses in the microgram per kilogram dose range (0.025 and 0.25 mg/kg bw per day). Time-mated Wistar rats (n=22) were gavaged during pregnancy and lactation from gestation day 7 to pup day 22 with 0, 0.025, 0.25, 5 or 50 mg/kg bw per day BPA. From 0.250 mg/kg and above, male anogenital distance (AGD) was significantly decreased, whereas decreased female AGD was seen from 0.025 mg/kg bw per day and above. Moreover, the incidence of nipple retention in males appeared to increase dose relatedly and the increase was statistically significant at 50 mg/kg per day. No significant changes in reproductive organ weights in the 16-day-old males and females and no signs of maternal toxicity were seen. The decreased AGD at birth in both sexes indicates effects on prenatal sexual development and provides new evidence of low-dose adverse effects of BPA in rats in the microgram per kilogram dose range. The NOAEL in this study is clearly below 5 mg/kg for BPA, which is used as the basis for establishment of the current tolerable daily intake (TDI) by EFSA; thus a reconsideration of the current TDI of BPA appears warranted.

Late-life effects on rat reproductive system after developmental exposure to mixtures of endocrine disrupters.

This study examined late-life effects of perinatal exposure of rats to a mixture of endocrine-disrupting contaminants. Four groups of 14 time-mated Wistar rats were exposed by gavage from gestation day 7 to pup day 22 to a mixture of 13 anti-androgenic and estrogenic chemicals including phthalates, pesticides, u.v.-filters, bisphenol A, parabens, and the drug paracetamol. The groups received vehicle (control), a mixture of all 13 chemicals at 150-times (TotalMix150) or 450-times (TotalMix450) high-end human exposure, or 450-times a mixture of nine predominantly anti-androgenic chemicals (AAMix450). Onset of puberty and estrous cyclicity at 9 and 12 months of age were assessed. Few female offspring showed significantly regular estrus cyclicity at 12 months of age in the TotalMix450 and AAMix450 groups compared with controls. In 19-month-old male offspring, epididymal sperm counts were lower than controls, and in ventral prostate an overrepresentation of findings related to hyperplasia was observed in exposed groups compared with controls, particularly in the group dosed with anti-androgens. A higher incidence of pituitary adenoma at 19 months of age was found in males and females in the AAMix450 group. Developmental exposure of rats to the highest dose of a human-relevant mixture of endocrine disrupters induced adverse effects late in life, manifested as earlier female reproductive senescence, reduced sperm counts, higher score for prostate atypical hyperplasia, and higher incidence of pituitary tumors. These delayed effects highlight the need for further studies on the role of endocrine disrupters in hormone-related disorders in aging humans.

Mixtures of endocrine-disrupting contaminants induce adverse developmental effects in preweaning rats.

Reproductive toxicity was investigated in rats after developmental exposure to a mixture of 13 endocrine-disrupting contaminants, including pesticides, plastic and cosmetic ingredients, and paracetamol. The mixture was composed on the basis of information about high-end human exposures, and the dose levels reflecting 100, 200, and 450 times this exposure were tested. The compounds were also grouped according to their estrogenicity or anti-androgenicity, and their joint effects were tested at two different doses, with each group reflecting 200 or 450 times human exposure. In addition, a single paracetamol dose was tested (350 mg/kg per day). All exposures and a vehicle were administered by oral gavage to time-mated Wistar dams rats throughout gestation and lactation, and their offspring were assessed for reproductive effects at birth and in prepuberty. The mixture doses, which included the anti-androgenic compounds, affected the male offspring by causing decreased anogenital distance, increased nipple retention (NR), and reduced ventral prostate weights, at both medium and high doses. In addition, the weights of the levator ani/bulbocavernosus muscle (LABC) were decreased at the high dose of anti-androgen mixture. No effects were seen after exposure to the estrogenic chemicals alone, whereas males exposed solely to paracetamol showed decreased LABC weights and increased NR. Thus adverse reproductive effects were observed at mixtures reflecting 200 times high-end human exposure, which is relatively close to the safety margin covered by the regulatory uncertainty factor of 100. This suggests that highly exposed human population groups may not be sufficiently protected against mixtures of endocrine-disrupting chemicals.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Intrauterine exposure to mild analgesics during pregnancy and the occurrence of cryptorchidism and hypospadia in the offspring: the Generation R Study.

BACKGROUND: Recently, over-the-counter mild analgesic use during pregnancy has been suggested to influence the risk of reproductive disorders in the offspring. We examined the influence of maternal exposure to mild analgesics during pregnancy on the occurrence of cryptorchidism and hypospadia in their offspring. METHODS: Associations between maternal exposure to mild analgesics during pregnancy and cryptorchidism or hypospadia in the offspring were studied in 3184 women participating in a large population-based prospective birth cohort study from early pregnancy onwards in the Netherlands (2002-2006), the Generation R Study. Cryptorchidism and hypospadia were identified during routine screening assessments performed in child health care centres by trained physicians. The use of mild analgesics was assessed in three prenatal questionnaires in pregnancy, resulting in four periods of use, namely, periconception period, first 14 weeks of gestation, 14-22 weeks of gestation and 20-32 weeks of gestation. Logistic regression analyses were used to study the associations between maternal exposure to mild analgesics and cryptorchidism and hypospadia. RESULTS: The cumulative prevalence over 30 months of follow up was 2.1% for cryptorchidism and 0.7% for hypospadia. Use of mild analgesics in the second period of pregnancy (14-22 weeks) increased the risk of congenital cryptorchidism [adjusted odds ratio (OR) 2.12; 95% confidence interval (CI) 1.17-3.83], primarily due to the use of acetaminophen (paracetamol) (adjusted OR 1.89; 95% CI 1.01-3.51). Among mothers of cryptorchid sons, 33.8% reported (23 of 68) the use of mild analgesics during pregnancy, compared with 31.8% (7 of 22) of mothers with a boy with hypospadia and 29.9% (926 of 3094) of mothers with healthy boys. CONCLUSIONS: Our results suggest that intrauterine exposure to mild analgesics, primarily paracetamol, during the period in pregnancy when male sexual differentiation takes place, increases the risk of cryptorchidism.

Developmental exposure to the DE-71 mixture of polybrominated diphenyl ether (PBDE) flame retardants induce a complex pattern of endocrine disrupting effects in rats.

Polybrominated diphenyl ethers (PBDEs) are legacy compounds with continued widespread human exposure. Despite this, developmental toxicity studies of DE-71, a mixture of PBDEs, are scarce and its potential for endocrine disrupting effects in vivo is not well covered. To address this knowledge gap, we carried out a developmental exposure study with DE-71. Pregnant Wistar rat dams were exposed to 0, 40 or 60 mg/kg bodyweight/day from gestation day 7 to postnatal day 16, and both sexes were examined. Developmental exposure affected a range of reproductive toxicity endpoints. Effects were seen for both male and female anogenital distances (AGD), with exposed offspring of either sex displaying around 10% shorter AGD compared to controls. Both absolute and relative prostate weights were markedly reduced in exposed male offspring, with about 40% relative to controls. DE-71 reduced mammary gland outgrowth, especially in male offspring. These developmental in vivo effects suggest a complex effect pattern involving anti-androgenic, anti-estrogenic and maybe estrogenic mechanisms depending on tissues and developmental stages. Irrespective of the specific underlying mechanisms, these in vivo results corroborate that DE-71 causes endocrine disrupting effects and raises concern for the effects of PBDE-exposure on human reproductive health, including any potential long-term consequences of disrupted mammary gland development.

Developmental exposure to the brominated flame retardant DE-71 reduces serum thyroid hormones in rats without hypothalamic-pituitary-thyroid axis activation or neurobehavioral changes in offspring.

Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants for which human exposure remains ubiquitous. This is of concern since these chemicals can perturb development and cause adverse health effects. For instance, DE-71, a technical mixture of PBDEs, can induce liver toxicity as well as reproductive and developmental toxicity. DE-71 can also disrupt the thyroid hormone (TH) system which may induce developmental neurotoxicity indirectly. However, in developmental toxicity studies, it remains unclear how DE-71 exposure affects the offspring's thyroid hormone system and if this dose-dependently relates to neurodevelopmental effects. To address this, we performed a rat toxicity study by exposing pregnant dams to DE-71 at 0, 40 or 60 mg/kg/day during perinatal development from gestational day 7 to postnatal day 16. We assessed the TH system in both dams and their offspring, as well as potential hearing and neurodevelopmental effects in prepubertal and adult offspring. DE-71 significantly reduced serum T4 and T3 levels in both dams and offspring without a concomitant upregulation of TSH, thus inducing a hypothyroxinemia-like effect. No discernible effects were observed on the offspring's brain function when assessed in motor activity boxes and in the Morris water maze, or on offspring hearing function. Our results, together with a thorough review of the literature, suggest that DE-71 does not elicit a clear dose-dependent relationship between low serum thyroxine (T4) and effects on the rat brain in standard behavioral assays. However, low serum TH levels are in themselves believed to be detrimental to human brain development, thus we propose that we lack assays to identify developmental neurotoxicity caused by chemicals disrupting the TH system through various mechanisms.

Using alternative test methods to predict endocrine disruption and reproductive adverse outcomes: do we have enough knowledge?

Endocrine disrupting chemicals (EDCs) are a matter of great concern. They are ubiquitous in the environment, are considered harmful to humans and wildlife, yet remain challenging to identify based on current international test guidelines and regulatory frameworks. For a compound to be identified as an EDC within the EU regulatory system, a plausible link between an endocrine mode-of-action and an adverse effect outcome in an intact organism must be established. This requires in-depth knowledge about molecular pathways regulating normal development and function in animals and humans in order to elucidate causes for disease. Although our knowledge about the role of the endocrine system in animal development and function is substantial, it remains challenging to predict endocrine-related disease outcomes in intact animals based on non-animal test data. A main reason for this is that our knowledge about mechanism-of-action are still lacking for essential causal components, coupled with the sizeable challenge of mimicking the complex multi-organ endocrine system by methodological reductionism. Herein, we highlight this challenge by drawing examples from male reproductive toxicity, which is an area that has been at the forefront of EDC research since its inception. We discuss the importance of increased focus on characterizing mechanism-of-action for EDC-induced adverse health effects. This is so we can design more robust and reliable testing strategies using non-animal test methods for predictive toxicology; both to improve chemical risk assessment in general, but also to allow for considerable reduction and replacement of animal experiments in chemicals testing of the 21st Century.

Intrauterine exposure to mild analgesics is a risk factor for development of male reproductive disorders in human and rat.

BACKGROUND: More than half of pregnant women in the Western world report intake of mild analgesics, and some of these drugs have been associated with anti-androgenic effects in animal experiments. Intrauterine exposure to anti-androgens is suspected to contribute to the recent increase in male reproductive problems, and many of the anti-androgenic compounds are like the mild analgesics potent inhibitors of prostaglandin synthesis. Therefore, it appears imperative to further investigate the potential endocrine disrupting properties of mild analgesics. METHODS: In a prospective birth cohort study, 2297 Danish and Finnish pregnant women completed a questionnaire and 491 of the Danish mothers participated in a telephone interview, reporting on their use of mild analgesics during pregnancy. The testicular position of newborns was assessed by trained paediatricians. In rats, the impact of mild analgesics on anogenital distance (AGD) after intrauterine exposure was examined together with the effect on ex vivo gestational day 14.5 testes. RESULTS: In the Danish birth cohort, the use of mild analgesics was dose-dependently associated with congenital cryptorchidism. In particular, use during the second trimester increased the risk. This risk was further increased after the simultaneous use of different analgesics. The association was not found in the Finnish birth cohort. Intrauterine exposure of rats to paracetamol led to a reduction in the AGD and mild analgesics accordingly reduced testosterone production in ex vivo fetal rat testes. CONCLUSION: There was an association between the timing and the duration of mild analgesic use during pregnancy and the risk of cryptorchidism. These findings were supported by anti-androgenic effects in rat models leading to impaired masculinization. Our results suggest that intrauterine exposure to mild analgesics is a risk factor for development of male reproductive disorders.

Effects of pre- and postnatal exposure to the UV-filter octyl methoxycinnamate (OMC) on the reproductive, auditory and neurological development of rat offspring.

Octyl Methoxycinnamate (OMC) is a frequently used UV-filter in sunscreens and other cosmetics. The aim of the present study was to address the potential endocrine disrupting properties of OMC, and to investigate how OMC induced changes in thyroid hormone levels would be related to the neurological development of treated offspring. Groups of 14-18 pregnant Wistar rats were dosed with 0, 500, 750 or 1000 mg OMC/kg bw/day during gestation and lactation. Serum thyroxine (T(4)), testosterone, estradiol and progesterone levels were measured in dams and offspring. Anogenital distance, nipple retention, postnatal growth and timing of sexual maturation were assessed. On postnatal day 16, gene expression in prostate and testes, and weight and histopathology of the thyroid gland, liver, adrenals, prostate, testes, epididymis and ovaries were measured. After weaning, offspring were evaluated in a battery of behavioral and neurophysiological tests, including tests of activity, startle response, cognitive and auditory function. In adult animals, reproductive organ weights and semen quality were investigated. Thyroxine (T(4)) levels showed a very marked decrease during the dosing period in all dosed dams, but were less severely affected in the offspring. On postnatal day 16, high dose male offspring showed reduced relative prostate and testis weights, and a dose-dependent decrease in testosterone levels. In OMC exposed female offspring, motor activity levels were decreased, while low and high dose males showed improved spatial learning abilities. The observed behavioral changes were probably not mediated solely by early T(4) deficiencies, as the observed effects differed from those seen in other studies of developmental hypothyroxinemia. At eight months of age, sperm counts were reduced in all three OMC-dosed groups, and prostate weights were reduced in the highest dose group. Taken together, these results indicate that perinatal OMC-exposure can affect both the reproductive and neurological development of rat offspring, which may be a cause of concern, as humans are systematically exposed to the compound through usage of sunscreens and other cosmetics.

On the impact of second generation mating and offspring in multi-generation reproductive toxicity studies on classification and labelling of substances in Europe.

The possible impact on classification and labelling decisions of effects observed in second generation parental (P1) and offspring (F2) parameters in multi-generation studies was investigated. This was done for 50 substances classified as reproductive toxicants in Europe, for which a multi-generation study was available. The P1 and F2 effects were compared to parental (P0) and first generation offspring (F1) effects with regard to type of effect as well as incidence, magnitude and severity (IMS), at any dose level. For every study with unique P1/F2 effects, or differences in IMS, the influence of the P1/F2 findings on the classification decision was investigated. Unique P1/F2 generation findings did not play a crucial role in the classification decision of any of the 50 classified substances, except for fenarimol. This substance however provided abundant alerts on the basis of its endocrine activity and developmental neurotoxicity and would therefore also be expected to be identified as a developmental neurotoxicant in an Extended One Generation Reproductive Toxicity Study (EOGRTS). These findings, in addition to the increased number of parameters analysed, increased statistical power and reduced animal use, provide strong further support for replacement of the classical two-generation reproductive toxicity study by the EOGRTS in regulatory reproductive toxicity assessment.

On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies.

Areola/nipple retention (NR) is an established biomarker for an anti-androgenic mode of action in rat toxicity studies. It is a mandatory measurement under several OECD test guidelines and is typically assessed in combination with anogenital distance (AGD). Both NR and AGD are considered retrospective biomarkers of insufficient androgen signaling during the masculinization programming window in male fetuses. However, there are still aspects concerning NR as a biomarker for endocrine disruption that remains to be clarified. For instance, can NR be regarded a permanent adverse effect? Is it a redundant measurement if AGD is assessed in the same study? Is NR equally sensitive and specific to anti-androgenic chemical substances as a shortening of male AGD? In this review we discuss these and other aspects concerning the use of NR as a biomarker in toxicity studies. We have collected available literature from rat toxicity studies that have reported on NR and synthesized the data in order to draw a clearer picture about the sensitivity and specificity of NR as an effect biomarker for an anti-androgenic mode of action, including comparisons to AGD measurements. We carefully conclude that NR and AGD in rats for the most part display similar sensitivity and specificity, but that there are clear exceptions which support the continued assessment of both endpoints in relevant reproductive toxicity studies. Available literature also support the view that NR in infant male rats signifies a high risk for permanent nipples in adulthood. Finally, the literature suggests that the mechanisms of action leading from a chemical stressor event to either NR or short AGD in male offspring are overlapping with respect to canonical androgen signaling, yet differ with respect to other mechanisms of action.

Evaluating thyroid hormone disruption: investigations of long-term neurodevelopmental effects in rats after perinatal exposure to perfluorohexane sulfonate (PFHxS).

Thyroid hormones are critical for mammalian brain development. Thus, chemicals that can affect thyroid hormone signaling during pregnancy are of great concern. Perfluorohexane sulfonate (PFHxS) is a widespread environmental contaminant found in human serum, breastmilk, and other tissues, capable of lowering serum thyroxine (T4) in rats. Here, we investigated its effects on the thyroid system and neurodevelopment following maternal exposure from early gestation through lactation (0.05, 5 or 25 mg/kg/day PFHxS), alone or in combination with a mixture of 12 environmentally relevant endocrine disrupting compounds (EDmix). PFHxS lowered thyroid hormone levels in both dams and offspring in a dose-dependent manner, but did not change TSH levels, weight, histology, or expression of marker genes of the thyroid gland. No evidence of thyroid hormone-mediated neurobehavioral disruption in offspring was observed. Since human brain development appear very sensitive to low T4 levels, we maintain that PFHxS is of potential concern to human health. It is our view that current rodent models are not sufficiently sensitive to detect adverse neurodevelopmental effects of maternal and perinatal hypothyroxinemia and that we need to develop more sensitive brain-based markers or measurable metrics of thyroid hormone-dependent perturbations in brain development.

Grouping of endocrine disrupting chemicals for mixture risk assessment - Evidence from a rat study.

Exposure to mixtures of endocrine disrupting chemicals may contribute to the rising incidence of hormone-related diseases in humans. Real-life mixtures are complex, comprised of chemicals with mixed modes of action, and essential knowledge is often lacking on how to group such chemicals into cumulative assessment groups, which is an essential prerequisite to conduct a chemical mixture risk assessment. We investigated if mixtures of chemicals with diverse endocrine modes of action can cause mixture effects on hormone sensitive endpoints in developing and adult rat offspring after perinatal exposure. Wistar rats were exposed during pregnancy and lactation simultaneously to either bisphenol A and butylparaben (Emix), diethylhexyl phthalate and procymidone (Amix), or a mixture of all four substances (Totalmix). In male offspring, the anogenital distance was significantly reduced and nipple retention increased in animals exposed to Amix and Totalmix, and the mixture effects were well approximated by the dose addition model. The combination of Amix and Emix responded with more marked changes on these and other endocrine-sensitive endpoints than each binary mixture on its own. Sperm counts were reduced by all exposures. These experimental outcomes suggest that the grouping of chemicals for mixture risk assessment should be based on common health outcomes rather than only similar modes or mechanisms of action. Mechanistic-based approaches such as the concept of Adverse Outcome Pathway (AOP) can provide important guidance if both the information on shared target tissues and the information on shared mode/mechanism of action are taken into account.

A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426.

OBJECTIVE: We conducted a review of the history and performance of developmental neurotoxicity (DNT) testing in support of the finalization and implementation of Organisation of Economic Co-operation and Development (OECD) DNT test guideline 426 (TG 426). INFORMATION SOURCES AND ANALYSIS: In this review we summarize extensive scientific efforts that form the foundation for this testing paradigm, including basic neurotoxicology research, interlaboratory collaborative studies, expert workshops, and validation studies, and we address the relevance, applicability, and use of the DNT study in risk assessment. CONCLUSIONS: The OECD DNT guideline represents the best available science for assessing the potential for DNT in human health risk assessment, and data generated with this protocol are relevant and reliable for the assessment of these end points. The test methods used have been subjected to an extensive history of international validation, peer review, and evaluation, which is contained in the public record. The reproducibility, reliability, and sensitivity of these methods have been demonstrated, using a wide variety of test substances, in accordance with OECD guidance on the validation and international acceptance of new or updated test methods for hazard characterization. Multiple independent, expert scientific peer reviews affirm these conclusions.