Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Comparison of female rat reproductive effects of pubertal versus adult exposure to known endocrine disruptors.

A prevailing challenge when testing chemicals for their potential to cause female reproductive toxicity is the lack of appropriate toxicological test methods. We hypothesized that starting a 28-day in vivo toxicity study already at weaning, instead of in adulthood, would increase the sensitivity to detect endocrine disruptors due to the possibility of including assessment of pubertal onset. We compared the sensitivity of two rat studies using pubertal or adult exposure. We exposed the rats to two well-known human endocrine disruptors, the estrogen diethylstilbestrol (DES; 0.003, 0.012, 0.048 mg/kg bw/day) and the steroid synthesis inhibitor ketoconazole (KTZ; 3, 12, 48 mg/kg bw/day). Specifically, we addressed the impact on established endocrine-sensitive endpoints including day of vaginal opening (VO), estrous cyclicity, weights of reproductive organs and ovarian histology. After 28 days of exposure, starting either at weaning or at 9 weeks of age, DES exposure altered estrous cyclicity, reduced ovary weight as well as number of antral follicles and corpora lutea. By starting exposure at weaning, we could detect advanced day of VO in DES-exposed animals despite a lower body weight. Some endpoints were affected mainly with adult exposure, as DES increased liver weights in adulthood only. For KTZ, no effects were seen on time of VO, but adrenal and liver weights were increased in both exposure scenarios, and adult KTZ exposure also stimulated ovarian follicle growth. At first glance, this would indicate that a pubertal exposure scenario would be preferrable as timing of VO may serve as sensitive indicator of endocrine disruption by estrogenic mode of action. However, a higher sensitivity for other endocrine targets may be seen starting exposure in adulthood. Overall, starting a 28-day study at weaning with inclusion of VO assessment would mainly be recommended for substances showing estrogenic potential e.g., in vitro, whereas for other substances an adult exposure scenario may be recommended.

Quantitative analysis of ovarian surface photographs as a tool for assessment of chemical effects on folliculogenesis and ovulation in rats.

Female reproductive toxicity assessments rely on histological evaluation of ovaries by hematoxylin & eosin (H&E)-stained cross-sections. This is time-consuming, labor-intensive and costly, thus alternative methods for ovarian toxicity assessment could be valuable. Here, we report on an improved method based on quantification of antral follicles (AF) and corpora lutea (CL) using ovarian surface photographs, called 'surface photo counting' (SPC). To validate a potential utility for the method to detect effects on folliculogenesis in toxicity studies, we investigated ovaries from rats exposed to two well-known endocrine disrupting chemicals (EDCs), diethylstilbestrol (DES) and ketoconazole (KTZ). Animals were exposed to DES (0.003, 0.012, 0.048 mg/kg body weight (bw)/day) or KTZ (3, 12, 48 mg/kg bw/day) during puberty or adulthood. At the end of the exposure, ovaries were photographed under stereomicroscope and subsequently processed for histological assessments to allow for direct comparison between the two methods by quantifying AF and CL. There was a significant correlation between the SPC and histology methods, albeit CL counts correlated better than AF counts, potentially due to their larger size. Effects of DES and KTZ were found by both methods, suggesting applicability of the SPC method to chemical hazard and risk assessment. Based on our study, we propose that SPC can be employed as a fast and cheap tool for assessment of ovarian toxicity in in vivo studies to prioritize chemical exposure groups for further histological assessment.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

Transcriptional profiling of the developing rat ovary following intrauterine exposure to the endocrine disruptors diethylstilbestrol and ketoconazole.

Exposure to endocrine-disrupting chemicals (EDCs) during development may cause reproductive disorders in women. Although female reproductive endpoints are assessed in rodent toxicity studies, a concern is that typical endpoints are not sensitive enough to detect chemicals of concern to human health. If so, measured endpoints must be improved or new biomarkers of effects included. Herein, we have characterized the dynamic transcriptional landscape of developing rat ovaries exposed to two well-known EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ), by 3' RNA sequencing. Rats were orally exposed from day 7 of gestation until birth, and from postnatal day 1 until days 6, 14 or 22. Three exposure doses for each chemical were used: 3, 6 and 12 µg/kg bw/day of DES; 3, 6, 12 mg/kg bw/day of KTZ. The transcriptome changed dynamically during perinatal development in control ovaries, with 1137 differentially expressed genes (DEGs) partitioned into 3 broad expression patterns. A cross-species deconvolution strategy based on a mouse ovary developmental cell atlas was used to map any changes to ovarian cellularity across the perinatal period to allow for characterization of actual changes to gene transcript levels. A total of 184 DEGs were observed across dose groups and developmental stages in DES-exposed ovaries, and 111 DEGs in KTZ-exposed ovaries across dose groups and developmental stages. Based on our analyses, we have identified new candidate biomarkers for female reproductive toxicity induced by EDC, including Kcne2, Calb2 and Insl3.

Exposure to the pesticides linuron, dimethomorph and imazalil alters steroid hormone profiles and gene expression in developing rat ovaries.

Inhibition of androgen signaling during critical stages of ovary development can disrupt folliculogenesis with potential consequences for reproductive function later in life. Many environmental chemicals can inhibit the androgen signaling pathway, which raises the question if developmental exposure to anti-androgenic chemicals can negatively impact female fertility. Here, we report on altered reproductive hormone profiles in prepubertal female rats following developmental exposure to three pesticides with anti-androgenic potential: linuron (25 and 50 mg/kg bw/d), dimethomorph (60 and 180 mg/kg bw/d) and imazalil (8 and 24 mg/kg bw/d). Dams were orally exposed from gestational day 7 (dimethomorph and imazalil) or 13 (linuron) until birth, then until end of dosing at early postnatal life. Linuron and dimethomorph induced dose-related reductions to plasma corticosterone levels, whereas imazalil mainly suppressed gonadotropin levels. In the ovaries, expression levels of target genes were affected by linuron and dimethomorph, suggesting impaired follicle growth. Based on our results, we propose that anti-androgenic chemicals can negatively impact female reproductive development. This highlights a need to integrate data from all levels of the hypothalamic-pituitary-gonadal axis, as well as the hypothalamic-pituitary-adrenal axis, when investigating the potential impact of endocrine disruptors on female reproductive development and function.

A Putative Adverse Outcome Pathway Network for Disrupted Female Pubertal Onset to Improve Testing and Regulation of Endocrine Disrupting Chemicals.

The average age for pubertal onset in girls has declined over recent decades. Epidemiological studies in humans and experimental studies in animals suggest a causal role for endocrine disrupting chemicals (EDCs) that are present in our environment. Of concern, current testing and screening regimens are inadequate in identifying EDCs that may affect pubertal maturation, not least because they do not consider early-life exposure. Also, the causal relationship between EDC exposure and pubertal timing is still a matter of debate. To address this issue, we have used current knowledge to elaborate a network of putative adverse outcome pathways (pAOPs) to identify how chemicals can affect pubertal onset. By using the AOP framework, we highlight current gaps in mechanistic understanding that need to be addressed and simultaneously point towards events causative of pubertal disturbance that could be exploited for alternative test methods. We propose 6 pAOPs that could explain the disruption of pubertal timing by interfering with the central hypothalamic trigger of puberty, GnRH neurons, and by so doing highlight specific modes of action that could be targeted for alternative test method development.

Perinatal exposure to known endocrine disrupters alters ovarian development and systemic steroid hormone profile in rats.

Disrupted ovarian development induced by chemical exposure may impair fertility later in life. Since androgens are essential for early ovarian development, we speculated that perinatal exposure to a binary mixture of the known anti-androgens DEHP and procymidone could alter steroid synthesis, disrupt ovarian follicle recruitment and ultimately maturation in female rat offspring. Wistar rat dams were exposed by oral gavage from gestation day 7 to postnatatal day 22 to two mixture doses known to alter reproductive development in male offspring (low: 10 mg/kg bw/day of procymidone and 30 mg/kg bw/day of DEHP; high: 20 mg/kg bw/day of procymidone and 60 mg/kg bw/day of DEHP). The Effects on plasma steroid hormones, ovarian follicle distribution and expression of markers related to steroid synthesis were examined in female offspring. In prepubertal offspring, we observed an increased number of newly recruited (primary) follicles in exposed animals compared to controls, and the plasma steroid hormone profile was altered by exposure: levels of progesterone, corticosterone and estrone were dose dependently elevated, whereas androgen levels were unaffected. In adulthood, a trend towards a smaller number of early-stage follicles may point to accelerated loss of follicle reserves, which is disconcerting. The changes in follicle distribution in exposed ovaries may reflect the combined influence of androgen receptor antagonism and altered ovarian steroid synthesis. This study adds to a growing body of evidence showing altered ovarian development following exposure to human relevant chemicals with possible severe consequences for female fertility.

Human-relevant concentrations of the antifungal drug clotrimazole disrupt maternal and fetal steroid hormone profiles in rats.

Clotrimazole is a non-prescription and broad-spectrum antifungal drug sold under brand names such as Canesten® and Lotrimin®. It is used to treat different types of fungal infections, from oral thrush to athlete's foot and vaginal mycosis. The level of exposure to clotrimazole is uncertain, as the exact usage amongst self-medicating patients is unclear. Recent studies have raised potential concern about the unsupervised use of clotrimazole during pregnancy, especially since it is a potent inhibitor of CYP enzymes of the steroidogenesis pathway. To address some of these concerns, we have assessed the effects of intrauterine exposure to clotrimazole on developing rat fetuses. By exposing pregnant rats to clotrimazole 25 or 75 mg/kg bw/day during gestation days 7-21, we obtained internal fetal concentrations close to those observed in humans. These in vivo data are in strong agreement with our physiologically-based pharmacokinetic (PBK)-modelled levels. At these doses, we observed no obvious morphological changes to the reproductive system, nor shorter male anogenital distance; a well-established morphometric marker for anti-androgenic effects in male offspring. However, steroid hormone profiles were significantly affected in both maternal and fetal plasma, in particular pronounced suppression of estrogens was seen. In fetal testes, marked up-concentration of hydroxyprogesterone was observed, which indicates a specific action on steroidogenesis. Since systemic clotrimazole is rapidly metabolized in humans, relevant exposure levels may not in itself cause adverse changes to the reproductive systems. Its capacity to significantly alter steroid hormone concentrations, however, suggests that clotrimazole should be used with caution during pregnancy.

Chemical Mixture Calculator - A novel tool for mixture risk assessment.

Humans are continuously exposed to complex chemical mixtures from foods and the environment. Experimental models in vivo and in vitro have increased our knowledge on how we can predict mixture effects. To accommodate a need for tools for efficient mixture risk assessment across different chemical classes and exposure sources, we have developed fit-for-purpose criteria for grouping of chemicals and a web-based tool for mixture risk assessment. The Chemical Mixture Calculator (available at www.chemicalmixturecalculator.dk) can be used for mixture risk assessment or identification of main drivers of risk. The underlying database includes hazard and exposure estimates for more than 200 chemicals in foods and environment. We present a range of cumulative assessment groups for effects on haematological system, kidney, liver, nervous system, developmental and reproductive system, and thyroid. These cumulative assessment groups are useful for grouping of chemicals at several levels of refinement depending on the question addressed. We present a mixture risk assessment case for phthalates, evaluated with and without contributions from other chemicals with similar effects. This case study shows the usefulness of the tool as a starting point for mixture risk assessment by the risk assessor, and emphasizes that solid scientific insight regarding underlying assumptions and uncertainties is crucial for result interpretation.

Classical toxicity endpoints in female rats are insensitive to the human endocrine disruptors diethylstilbestrol and ketoconazole.

Developmental exposure to endocrine disrupting chemicals can have negative consequences for reproductive health in both men and women. Our knowledge about how chemicals can cause adverse health outcomes in females is, however, poorer than our knowledge in males. This is possibly due to lack of sensitive endpoints to evaluate endocrine disruption potential in toxicity studies. To address this shortcoming we carried out rat studies with two well-known human endocrine disruptors, diethylstilbestrol (DES) and ketoconazole (KTZ), and evaluated the sensitivity of a series of endocrine related endpoints. Sprague-Dawley rats were exposed orally from gestational day 7 until postnatal day 22. In a range-finding study, disruption of pregnancy-related endpoints was seen from 0.014 mg/kg bw/day for DES and 14 mg/kg bw/day for KTZ, so doses were adjusted to 0.003; 0.006; and 0.0012 mg/kg bw/day DES and 3; 6; or 12 mg/kg bw/day KTZ in the main study. We observed endocrine disrupting effects on sensitive endpoints in male offspring: both DES and KTZ shortened anogenital distance and increased nipple retention. In female offspring, 0.0012 mg/kg bw/day DES caused slightly longer anogenital distance. We did not see effects on puberty onset when comparing average day of vaginal opening; however, we saw a subtle delay after exposure to both chemicals using a time-curve analysis. No effects on estrous cycle were registered. Our study shows a need for more sensitive test methods to protect the reproductive health of girls and women from harmful chemicals.

Transcriptome analysis of fetal rat testis following intrauterine exposure to the azole fungicides triticonazole and flusilazole reveals subtle changes despite adverse endocrine effects.

Azoles are used in agriculture and medicine to combat fungal infections. We have previously examined the endocrine disrupting properties of the agricultural azole fungicides triticonazole and flusilazole. Triticonazole displayed strong androgen receptor (AR) antagonism in vitro, whereas in utero exposure resulted in anti-androgenic effects in vivo evidenced by shorter anogenital distance (AGD) in fetal male rats. Flusilazole displayed strong AR antagonism, but less potent than triticonazole, and disrupted steroidogenesis in vitro, whereas in utero exposure disrupted fetal male plasma hormone levels. To elaborate on how these azole fungicides can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes, we profiled fetal testis transcriptomes after in utero exposure to triticonazole and flusilazole by 3'Digital Gene Expression (3'DGE). The analysis revealed few transcriptional changes after exposure to either compound at gestation day 17 and 21. This suggests that the observed influence of flusilazole on hormone production may be by directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas observations of shorter AGD by triticonazole may primarily be due to disturbed androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes.

Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders.

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (λ-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Using assessment criteria for pesticides to evaluate the endocrine disrupting potential of non-pesticide chemicals: Case butylparaben.

Regulation of chemicals with endocrine disrupting properties depend on the use of the chemical rather than its intrinsic properties. Within the EU, the only criteria currently in place for identifying an endocrine disrupting chemical (EDC) are those developed for biocidal and plant protection products. We argue that ECHA/EFSA guidance for assessing endocrine disrupting properties of biocidal and plant protection products can be applied to all chemicals independent of their intended use. We have assessed the REACH-registered compound butylparaben (CAS 94-36-8), a preservative used primarily in cosmetics. Based on scientific evidence of adverse reproductive effects and endocrine activity, the open literature suggest that butylparaben is an EDC. By applying the ECHA/EFSA guidance for pesticides and biocides, we identify butylparaben as a compound with endocrine disrupting properties. Even though available data is markedly different from that for biocides and pesticides, it was possible to reach this conclusion. More generally, we propose that the ECHA/EFSA guidance can and should be used for identification of EDC regardless of their intended application.

Putative adverse outcome pathways for female reproductive disorders to improve testing and regulation of chemicals.

Modern living challenges female reproductive health. We are witnessing a rise in reproductive disorders and drop in birth rates across the world. The reasons for these manifestations are multifaceted and most likely include continuous exposure to an ever-increasing number of chemicals. The cause-effect relationships between chemical exposure and female reproductive disorders, however, have proven problematic to determine. This has made it difficult to assess the risks chemical exposures pose to a woman's reproductive development and function. To address this challenge, this review uses the adverse outcome pathway (AOP) concept to summarize current knowledge about how chemical exposure can affect female reproductive health. We have a special focus on effects on the ovaries, since they are essential for lifelong reproductive health in women, being the source of both oocytes and several reproductive hormones, including sex steroids. The AOP framework is widely accepted as a new tool for toxicological safety assessment that enables better use of mechanistic knowledge for regulatory purposes. AOPs equip assessors and regulators with a pragmatic network of linear cause-effect relationships, enabling the use of a wider range of test method data in chemical risk assessment and regulation. Based on current knowledge, we propose ten putative AOPs relevant for female reproductive disorders that can be further elaborated and potentially be included in the AOPwiki. This effort is an important step towards better safeguarding the reproductive health of all girls and women.

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.

Safeguarding Female Reproductive Health against Endocrine Disrupting Chemicals-The FREIA Project.

Currently available test methods are not well-suited for the identification of chemicals that disturb hormonal processes involved in female reproductive development and function. This renders women's reproductive health at increasing risk globally, which, coupled with increasing incidence rates of reproductive disorders, is of great concern. A woman's reproductive health is largely established during embryonic and fetal development and subsequently matures during puberty. The endocrine system influences development, maturation, and function of the female reproductive system, thereby making appropriate hormone levels imperative for correct functioning of reproductive processes. It is concerning that the effects of human-made chemicals on the endocrine system and female reproductive health are poorly addressed in regulatory chemical safety assessment, partly because adequate test methods are lacking. Our EU-funded project FREIA aims to address this need by increasing understanding of how endocrine disrupting chemicals (EDCs) can impact female reproductive health. We will use this information to provide better test methods that enable fit-for-purpose chemical regulation and then share our knowledge, promote a sustainable society, and improve the reproductive health of women globally.

Dietary exposure to selected chemical contaminants in fish for the Danish population.

The exposure to selected chemical contaminants from fish has been calculated for the Danish population, both for adults (15-75 years of age) and children (4-14 years of age). The Danish mean consumption of fish is 21 g person-1 day-1 for adults and 12 g person-1 day-1 for children. Fish consumption is the main food group contributor for exposure to mercury and dioxins and dioxin-like polychlorinated biphenyls (PCDD/F and DL-PCB) for the Danish population. Comparison of the mean exposure with the TDI or TWI values shows for these substances as well as for perfluorooctane sulphuric acid (PFOS) that the exposure is below the TDI/TWI values. However, even without taking other food groups into account, PCDD/Fs and DL-PCB exposure is close to the actual TWI-value. Calculation of the Margin of Exposure (MOE) for the sum of hexabromocyclododecanes (HBCDD) and polycyclic aromatic hydrocarbons (PAHs) revealed fish consumption to be of low concern for the consumer health regarding these contaminants.

Calretinin is a novel candidate marker for adverse ovarian effects of early life exposure to mixtures of endocrine disruptors in the rat.

Disruption of sensitive stages of ovary development during fetal and perinatal life can have severe and life-long consequences for a woman's reproductive life. Exposure to endocrine disrupting chemicals may affect ovarian development, leading to subsequent reproductive disorders. Here, we investigated the effect of early life exposure to defined mixtures of human-relevant endocrine disrupting chemicals on the rat ovary. We aimed to identify molecular events involved in pathogenesis of ovarian dysgenesis syndrome that have potential for future adverse outcome pathway development. We therefore focused on the ovarian proteome. Rats were exposed to a mixture of phthalates, pesticides, UV-filters, bisphenol A, butyl-paraben, and paracetamol during gestation and lactation. The chemicals were tested together or in subgroups of chemicals with anti-androgenic or estrogenic potentials at doses 450-times human exposure. Paracetamol was tested separately, at a dose of 360 mg/kg. Using shotgun proteomics on ovaries from pup day 17 offspring, we observed exposure effects on the proteomes. Nine proteins were affected in more than one exposure group and of these, we conclude that calretinin is a potential key event biomarker of early endocrine disruption in the ovary.

Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways.

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.