M-FISH evaluation of chromosome aberrations to examine for historical exposure to ionising radiation due to participation at British nuclear test sites.

Veterans of the British nuclear testing programme represent a population of ex-military personnel who had the potential to be exposed to ionising radiation through their participation at nuclear testing sites in the 1950s and 1960s. In the intervening years, members of this population have raised concerns about the status of their health and that of their descendants, as a consequence. Radiation dose estimates based on film badge measurements of external dose recorded at the time of the tests suggest any exposure to be limited for the majority of personnel, however, only ∼20% of personnel were monitored and no measurement for internalised exposure are on record. Here, to in-part address families concerns, we assay for chromosomal evidence of historical radiation exposure in a group of aged nuclear test (NT) veterans, using multiplexin situhybridisation (M-FISH), for comparison with a matched group of veterans who were not present at NT sites. In total, we analysed 9379 and 7698 metaphase cells using M-FISH (24-colour karyotyping) from 48 NT and 38 control veteran samples, representing veteran servicemen from the army, Royal Airforce and Royal Navy. We observed stable and unstable simple- and complex-type chromosome aberrations in both NT and control veterans' samples, however find no significant difference in yield of any chromosome aberration type between the two cohorts. We do observe higher average frequencies of complex chromosome aberrations in a very small subset of veterans previously identified as having a higher potential for radiation exposure, which may be indicative of internalised contamination to long-lived radionuclides from radiation fallout. By utilising recently published whole genome sequence analysis data of a sub-set of the same family groups, we examined for but found no relationship between paternal chromosome aberration burden, germline mutation frequency and self-reported concerns of adverse health in family members, suggesting that the previously reported health issues by participants in this study are unlikely to be associated with historical radiation exposure. We did observe a small number of families, representing both control and NT cohorts, showing a relationship between paternal chromosome aberrations and germline mutation sub-types which should be explored in future studies. In conclusion, we find no cytogenetic evidence of historical radiation exposure in the cohort of nuclear veterans sampled here, offering reassurance that attendance at NTs sites by the veterans sampled here, was not associated with significant levels of exposure to radiation.

Screening for endocrine disrupting chemicals inhibiting monocarboxylate 8 (MCT8) transporter facilitated thyroid hormone transport using a modified nonradioactive assay.

Early neurodevelopmental processes are strictly dependent on spatial and temporally modulated of thyroid hormone (TH) availability and action. Thyroid hormone transmembrane transporters (THTMT) are critical for regulating the local concentrations of TH, namely thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3), in the brain. Monocarboxylate transporter 8 (MCT8) is one of the most prominent THTMT. Genetically induced deficiencies in expression, function or localization of MCT8 are associated with irreversible and severe neurodevelopmental adversities. Due to the importance of MCT8 in brain development, studies addressing chemical interferences of MCT8 facilitated T3 uptake are a crucial step to identify TH system disrupting chemicals with this specific mode of action. Recently a non-radioactive in vitro assay has been developed to rapidly screen for endocrine disrupting chemicals (EDCs) acting upon MCT8 mediated transport. This study explored the use of an UV-light digestion step as an alternative for the original ammonium persulfate (APS) digestion step. The non-radioactive TH uptake assay, with the incorporated UV-light digestion step of TH, was then used to screen a set of 31 reference chemicals and environmentally relevant substances to detect inhibition of MCT8-depending T3 uptake. This alternative assay identified three novel MCT8 inhibitors: methylmercury, bisphenol-AF and bisphenol-Z and confirmed previously known MCT8 inhibitors.

The impact of biostatistics on hazard characterization using in vitro developmental neurotoxicity assays.

In chemical safety assessment, benchmark concentrations (BMC) and their associated uncertainty are needed for the toxicological evaluation of in vitro data sets. A BMC estimation is derived from concentration-response modelling and results from various statistical decisions, which depend on factors such as experimental design and assay endpoint features. In current data practice, the experimenter is often responsible for the data analysis and therefore relies on statistical software, often without being aware of the software default settings and how they can impact the outputs of data analysis. To provide more insight into how statistical decision-making can influence the outcomes of data analysis and interpretation, we have developed an automated platform that includes statistical methods for BMC estimation, a novel endpoint-specific hazard classification system, and routines that flag data sets that are outside the applicability domain for an automatic data evaluation. We used case studies on a large dataset produced by a developmental neurotoxicity (DNT) in vitro battery (DNT IVB). Here we focused on the BMC and its confidence interval (CI) estimation as well as on final hazard classification. We identified five crucial statistical decisions the experimenter must make during data analysis: choice of replicate averaging, response data normalization, regression modelling, BMC and CI estimation, and choice of benchmark response levels. The insights gained are intended to raise more awareness among experimenters on the importance of statistical decisions and methods but also to demonstrate how important fit-for-purpose, internationally harmonized and accepted data evaluation and analysis procedures are for objective hazard classification.

Establishment of a human cell-based in vitro battery to assess developmental neurotoxicity hazard of chemicals.

Developmental neurotoxicity (DNT) is a major safety concern for all chemicals of the human exposome. However, DNT data from animal studies are available for only a small percentage of manufactured compounds. Test methods with a higher throughput than current regulatory guideline methods, and with improved human relevance are urgently needed. We therefore explored the feasibility of DNT hazard assessment based on new approach methods (NAMs). An in vitro battery (IVB) was assembled from ten individual NAMs that had been developed during the past years to probe effects of chemicals on various fundamental neurodevelopmental processes. All assays used human neural cells at different developmental stages. This allowed us to assess disturbances of: (i) proliferation of neural progenitor cells (NPC); (ii) migration of neural crest cells, radial glia cells, neurons and oligodendrocytes; (iii) differentiation of NPC into neurons and oligodendrocytes; and (iv) neurite outgrowth of peripheral and central neurons. In parallel, cytotoxicity measures were obtained. The feasibility of concentration-dependent screening and of a reliable biostatistical processing of the complex multi-dimensional data was explored with a set of 120 test compounds, containing subsets of pre-defined positive and negative DNT compounds. The battery provided alerts (hit or borderline) for 24 of 28 known toxicants (82% sensitivity), and for none of the 17 negative controls. Based on the results from this screen project, strategies were developed on how IVB data may be used in the context of risk assessment scenarios employing integrated approaches for testing and assessment (IATA).

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

No evidence of increased mutations in the germline of a group of British nuclear test veterans.

The potential germline effects of radiation exposure to military veterans present at British nuclear tests in Australia and the South Pacific is of considerable interest. We analyzed germline mutations in 60 families of UK military personnel comprising 30 control and 30 nuclear test veterans (NTV). Using whole-genome sequencing we studied the frequency and spectra of de novo mutations to investigate the transgenerational effect of veterans' (potential) exposure to radiation at nuclear bomb test sites. We find no elevation in total de novo single nucleotide variants, small insertion-deletions, structural variants or clustered mutations among the offspring of nuclear test veterans compared to those of control personnel. We did observe an elevated occurrence of single base substitution mutations within mutation signature SBS16, due to a subset of NTV offspring. The relevance of this elevation to potential exposure of veteran fathers and, future health risks, require further investigation. Overall, we find no evidence of increased mutations in the germline of a group of British nuclear test veterans. ISRCTN Registry 17461668.

Combined exposures to bisphenols, polychlorinated dioxins, paracetamol, and phthalates as drivers of deteriorating semen quality.

BACKGROUND: Semen quality in men continues to decline in Western countries, but the contours of the issue remain obscure, in relation to contributing chemicals. OBJECTIVES: To obtain more clarity about the chemicals that drive the deterioration of semen quality, we conducted a mixture risk assessment based on European exposures. METHODS: We included chemicals capable of affecting semen quality after prenatal exposures, among them androgen receptor antagonists, substances that disrupt prostaglandin signalling, suppress testosterone synthesis, inhibit steroidogenic enzymes or activate the aryl hydrocarbon receptor. We employed the Hazard Index approach (HI), based on risk quotients of exposures in Europe and reference doses for reductions in semen quality. By summing up the risk quotients of the 29 chemicals included in the assessment we examined fold-exceedances of "acceptable" mixture exposures relative to an index value of 1. For bisphenols A, F, S, phthalates DEHP, DnBP, BBzP, DiNP, n-butyl paraben and paracetamol we relied on biomonitoring studies in which these 9 chemicals were measured together in the same subjects. This allowed us to construct personalised Hazard Indices. RESULTS: Highly exposed subjects experienced combined exposures to the 9 chemicals that exceeded the index value of 1 by more than 100-fold; the median was a 17-fold exceedance. Accounting for median background exposures to the remaining 20 chemicals added a Hazard Index of 1.39. Bisphenol A made the largest contribution to the HI, followed by polychlorinated dioxins, bisphenols S and F and DEHP. Eliminating bisphenol A alone would still leave unacceptably high mixture risks. Paracetamol is also a driver of mixture risks among subjects using the drug. CONCLUSIONS: Tolerable exposures to substances associated with deteriorations of semen quality are exceeded by a large margin. Bisphenols, polychlorinated dioxins, phthalates and analgesics drive these risks. Dedicated efforts towards lowering exposures to these substances are necessary to mitigate risks.

British nuclear test veteran family trios for the study of genetic risk.

The risk of radiation effects in children of individuals exposed to ionising radiation remains an ongoing concern for aged veterans of the British nuclear testing programme. The genetic and cytogenetic family trio (GCFT) study is the first study to obtain blood samples from a group of British nuclear test veterans and their families for the purposes of identifying genetic alterations in offspring as a consequence of historical paternal exposure to ionising radiation. In this report, we describe the processes for recruitment and sampling, and provide a general description of the study population recruited. In total, blood samples were received from 91 (49 test and 42 control) families representing veteran servicemen from the army, Royal Air Force and Royal Navy. This translated to an overall response rate of 14% (49/353) for test veterans and 4% (42/992) for control veterans (excluding responders known to be ineligible). Due to the lack of dose information available, test veterans were allocated to a three-point exposure rank. Thirty (61%) test veterans were ranked in the lower group. Nineteen (39%) of the 49 test veterans were classified in the mid (5 veterans; 10%)/high (14 veterans; 29%) exposure ranks and included 12 veterans previously identified as belonging to the special groups or listed in health physics documents. An increased number of test veteran families (20%), compared with control families (5%), self-reported offspring with congenital abnormalities (p= 0.03). Whether this observation in this small group is reflective of the entire UK test veteran cohort or whether it is selection bias requires further work. The cohort described here represent an important and unique family trio grouping whose participation is enabling genetic studies, as part of the GCFT study, to be carried out. The outcomes of these studies will be published elsewhere. ISRCTN Registry: 17461668.

Bisphenol A and declining semen quality: A systematic review to support the derivation of a reference dose for mixture risk assessments.

To support a mixture risk assessment with a focus on male reproductive health, we conducted a systematic review of associations between bisphenol A (BPA) exposures and declines in semen quality, based on animal and epidemiological studies. Contrary to a widely held view that there is "conflicting" evidence of such associations, our review and confidence rating approach reveals that animal studies provide convincing evidence of declines of semen quality after gestational BPA exposures. Many of the reported negative findings can be attributed to deficiencies in study sensitivity, insufficient control of background contamination and probable confounding through hormonal interference due to the use of soy-containing diets. We did not evaluate animal studies of adult BPA exposures. Divergent findings in "medium to high" and "medium" confidence epidemiological studies can be explained in terms of differences in exposure conditions. We attempted the estimation of a BPA reference dose based on animal studies. Due to variations in the no-observed adverse effect levels (NOAELs) in high confidence studies, possible reference doses ranged from 0.0001 to 0.0099 µg/kg/d. In choosing 0.003 µg/kg/d we struck a balance between caution suggested by studies at the lower end of the doses and the weight of evidence from studies with higher NOAELs. This weighting was motivated by the intended use of the value in a mixture risk assessment which meant arriving at a reasonable estimate of BPA exposures likely without effects on semen quality. We realise that our approach does not conform with the standards necessary for deriving tolerable daily intakes (TDIs) for single chemical exposures, which is not our interest here. BPA exposures currently experienced by European populations and beyond are in excess of 0.003 µg/kg/d and even fall in the range where some epidemiological studies observed effects on semen quality as a result of BPA exposures in adulthood.

Testing for heterotopia formation in rats after developmental exposure to selected in vitro inhibitors of thyroperoxidase.

The thyroperoxidase (TPO) enzyme is expressed by the thyroid follicular cells and is required for thyroid hormone synthesis. In turn, thyroid hormones are essential for brain development, thus inhibition of TPO in early life can have life-long consequences for brain function. If environmental chemicals with the capacity to inhibit TPO in vitro can also alter brain development in vivo through thyroid hormone dependent mechanisms, however, remains unknown. In this study we show that the in vitro TPO inhibiting pesticide amitrole alters neuronal migration and induces periventricular heterotopia; a thyroid hormone dependent brain malformation. Perinatal exposure to amitrole reduced pup serum thyroxine (T4) concentrations to less than 50% of control animals and this insufficiency led to heterotopia formation in the 16-day old pup's brain. Two other in vitro TPO inhibitors, 2-mercaptobenzimidazole and cyanamide, caused reproductive toxicity and had only minor sporadic effects on the thyroid hormone system; consequently, they did not cause heterotopia. This is the first demonstration of an environmental chemical causing heterotopia, a brain malformation until now only reported for rodent studies with the anti-thyroid drugs propylthiouracil and methimazole. Our results highlight that certain TPO-inhibiting environmental chemicals can alter brain development through thyroid hormone dependent mechanisms. Improved understanding of the effects on the brain as well as the conditions under which chemicals can perturb brain development will be key to protect human health.

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.

Ten years of research on synergisms and antagonisms in chemical mixtures: A systematic review and quantitative reappraisal of mixture studies.

BACKGROUND: Several reviews of synergisms and antagonisms in chemical mixtures have concluded that synergisms are relatively rare. However, these reviews focused on mixtures composed of specific groups of chemicals, such as pesticides or metals and on toxicity endpoints mostly relevant to ecotoxicology. Doubts remain whether these findings can be generalised. A systematic review not restricted to specific chemical mixtures and including mammalian and human toxicity endpoints is missing. OBJECTIVES: We conducted a systematic review and quantitative reappraisal of 10 years' of experimental mixture studies to investigate the frequency and reliability of evaluations of mixture effects as synergistic or antagonistic. Unlike previous reviews, we did not limit our efforts to certain groups of chemicals or specific toxicity outcomes and covered mixture studies relevant to ecotoxicology and human/mammalian toxicology published between 2007 and 2017. DATA SOURCES, ELIGIBILITY CRITERIA: We undertook searches for peer-reviewed articles in PubMed, Web of Science, Scopus, GreenFile, ScienceDirect and Toxline and included studies of controlled exposures of environmental chemical pollutants, defined as unintentional exposures leading to unintended effects. Studies with viruses, prions or therapeutic agents were excluded, as were records with missing details on chemicals' identities, toxicities, doses, or concentrations. STUDY APPRAISAL AND SYNTHESIS METHODS: To examine the internal validity of studies we developed a risk-of-bias tool tailored to mixture toxicology. For a subset of 388 entries that claimed synergisms or antagonisms, we conducted a quantitative reappraisal of authors' evaluations by deriving ratios of predicted and observed effective mixture doses (concentrations). RESULTS: Our searches produced an inventory of 1220 mixture experiments which we subjected to subgroup analyses. Approximately two thirds of studies did not incorporate more than 2 components. Most experiments relied on low-cost assays with readily quantifiable endpoints. Important toxicity outcomes of relevance for human risk assessment (e.g. carcinogenicity, genotoxicity, reproductive toxicity, immunotoxicity, neurotoxicity) were rarely addressed. The proportion of studies that declared additivity, synergism or antagonisms was approximately equal (one quarter each); the remaining quarter arrived at different evaluations. About half of the 1220 entries were rated as "definitely" or "probably" low risk of bias. Strikingly, relatively few claims of synergistic or antagonistic effects stood up to scrutiny in terms of deviations from expected additivity that exceed the boundaries of acceptable between-study variability. In most cases, the observed mixture doses were not more than two-fold higher or lower than the predicted additive doses. Twenty percent of the entries (N = 78) reported synergisms in excess of that degree of deviation. Our efforts of pinpointing specific factors that predispose to synergistic interactions confirmed previous concerns about the synergistic potential of combinations of triazine, azole and pyrethroid pesticides at environmentally relevant doses. New evidence of synergisms with endocrine disrupting chemicals and metal compounds such as chromium (VI) and nickel in combination with cadmium has emerged. CONCLUSIONS, LIMITATIONS AND IMPLICATIONS: These specific cases of synergisms apart, our results confirm the utility of default application of the dose (concentration) addition concept for predictive assessments of simultaneous exposures to multiple chemicals. However, this strategy must be complemented by an awareness of the synergistic potential of specific classes of chemicals. Our conclusions only apply to the chemical space captured in published mixture studies which is biased towards relatively well-researched chemicals. SYSTEMATIC REVIEW REGISTRATION NUMBER: The final protocol was published on the open-access repository Zenodo and attributed the following digital object identifier, doi: https://doi.org//10.5281/zenodo.1319759 (https://zenodo.org/record/1319759#.XXIzdy7dsqM).

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.

The ENDpoiNTs Project: Novel Testing Strategies for Endocrine Disruptors Linked to Developmental Neurotoxicity.

Ubiquitous exposure to endocrine-disrupting chemicals (EDCs) has caused serious concerns about the ability of these chemicals to affect neurodevelopment, among others. Since endocrine disruption (ED)-induced developmental neurotoxicity (DNT) is hardly covered by the chemical testing tools that are currently in regulatory use, the Horizon 2020 research and innovation action ENDpoiNTs has been launched to fill the scientific and methodological gaps related to the assessment of this type of chemical toxicity. The ENDpoiNTs project will generate new knowledge about ED-induced DNT and aims to develop and improve in vitro, in vivo, and in silico models pertaining to ED-linked DNT outcomes for chemical testing. This will be achieved by establishing correlative and causal links between known and novel neurodevelopmental endpoints and endocrine pathways through integration of molecular, cellular, and organismal data from in vitro and in vivo models. Based on this knowledge, the project aims to provide adverse outcome pathways (AOPs) for ED-induced DNT and to develop and integrate new testing tools with high relevance for human health into European and international regulatory frameworks.

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.

Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals-The ATHENA Project.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

Transthyretin-Binding Activity of Complex Mixtures Representing the Composition of Thyroid-Hormone Disrupting Contaminants in House Dust and Human Serum.

BACKGROUND: House dust contains many organic contaminants that can compete with the thyroid hormone (TH) thyroxine (T4) for binding to transthyretin (TTR). How these contaminants work together at levels found in humans and how displacement from TTR in vitro relates to in vivo T4-TTR binding is unknown. OBJECTIVES: Our aims were to determine the TTR-binding potency for contaminant mixtures as found in house dust, maternal serum, and infant serum; to study whether the TTR-binding potency of the mixtures follows the principle of concentration addition; and to extrapolate the in vitro TTR-binding potency to in vivo inhibition levels of T4-TTR binding in maternal and infant serum. METHODS: Twenty-five contaminants were tested for their in vitro capacity to compete for TTR-binding with a fluorescent FITC-T4 probe. Three mixtures were reconstituted proportionally to median concentrations for these chemicals in house dust, maternal serum, or infant serum from Nordic countries. Measured concentration-response curves were compared with concentration-response curves predicted by concentration addition. For each reconstituted serum mixture, its inhibitor-TTR dissociation constant (Ki) was used to estimate inhibition levels of T4-TTR binding in human blood. RESULTS: The TTR-binding potency of the mixtures was well predicted by concentration addition. The ∼20% inhibition in FITC-T4 binding observed for the mixtures reflecting median concentrations in maternal and infant serum was extrapolated to 1.3% inhibition of T4-TTR binding in maternal and 1.5% in infant blood. For nontested mixtures reflecting high-end serum concentrations, these estimates were 6.2% and 4.9%, respectively. DISCUSSION: The relatively low estimated inhibition levels at median exposure levels may explain why no relationship between exposure to TTR-binding compounds and circulating T4 levels in humans has been reported, so far. We hypothesize, however, that 1.3% inhibition of T4-TTR binding may ultimately be decisive for reaching a status of maternal hypothyroidism or hypothyroxinemia associated with impaired neurodevelopment in children. https://doi.org/10.1289/EHP5911.

Combined effects of environmental xeno-estrogens within multi-component mixtures: Comparison of in vitro human- and zebrafish-based estrogenicity bioassays.

Some recent studies showed that in vitro bioassays based on fish or human estrogen receptor (ER) activation may have distinct responses to environmental samples, highlighting the need to better understand bioassay-specific ER response to environmental mixtures. For this purpose, we investigated a 12-compound mixture in two mixture ratios (M1 and M2) on zebrafish (zf) liver cells stably expressing zfERα (ZELHα cells) or zfERß2 (ZELHß2 cells) and on human ER-reporter gene (MELN) cells. The mixture included the well-known ER ligands bisphenol A (BPA) and genistein (GEN), and other compounds representatives of a freshwater background contamination. In this context, the study aimed at assessing the robustness of concentration addition (CA) model and the potential confounding influence of other chemicals by testing subgroups of ER activators, ER inhibitors or ER activators and inhibitors combined. Individual chemical testing showed a higher prevalence of ER inhibitors in zebrafish than human cells (e.g. propiconazole), and some chemicals inhibited zfER but activated hER response (e.g. benzo(a)pyrene, triphenylphosphate). The estrogenic activity of M1 and M2 was well predicted by CA in MELN cells, whereas it was significantly lower than predicted in ZELHß2 cells, contrasting with the additive effects observed for BPA and GEN binary mixtures. When testing the subgroups of ER activators and inhibitors combined, the deviation from additivity in ZELHß2 cells was caused by zebrafish-specific inhibiting chemicals. This study provides novel information on the ability of environmental pollutants to interfere with zfER signalling and shows that non-estrogenic chemicals can influence the response to a mixture of xeno-estrogens in a bioassay-specific manner.

The consequences of exposure to mixtures of chemicals: Something from 'nothing' and 'a lot from a little' when fish are exposed to steroid hormones.

Ill-defined, multi-component mixtures of steroidal pharmaceuticals are present in the aquatic environment. Fish are extremely sensitive to some of these steroids. It is important to know how fish respond to these mixtures, and from that knowledge develop methodology that enables accurate prediction of those responses. To provide some of the data required to reach this objective, pairs of fish were first exposed to five different synthetic steroidal pharmaceuticals (one estrogen, EE2; one androgen, trenbolone; one glucocorticoid, beclomethasone dipropionate; and two progestogens, desogestrel and levonorgestrel) and concentration-response data on egg production obtained. Based on those concentration-response relationships, a five component mixture was designed and tested twice. Very similar effects were observed in the two experiments. The mixture inhibited egg production in an additive manner predicted better by the model of Independent Action than that of Concentration Addition. Our data provide a reference case for independent action in an in vivo model. A significant combined effect was observed when each steroidal pharmaceutical in the mixture was present at a concentration which on its own would produce no statistically significant effect (something from 'nothing'). Further, when each component was present in the mixture at a concentration expected to inhibit egg production by between 18% (Beclomethasone diproprionate) and 40% (trenbolone), this mixture almost completely inhibited egg production: a phenomenon we term 'a lot from a little'. The results from this proof-of-principle study suggest that multiple steroids present in the aquatic environment can be analysed for their potential combined environmental risk.

Mixture effects in samples of multiple contaminants - An inter-laboratory study with manifold bioassays.

Chemicals in the environment occur in mixtures rather than as individual entities. Environmental quality monitoring thus faces the challenge to comprehensively assess a multitude of contaminants and potential adverse effects. Effect-based methods have been suggested as complements to chemical analytical characterisation of complex pollution patterns. The regularly observed discrepancy between chemical and biological assessments of adverse effects due to contaminants in the field may be either due to unidentified contaminants or result from interactions of compounds in mixtures. Here, we present an interlaboratory study where individual compounds and their mixtures were investigated by extensive concentration-effect analysis using 19 different bioassays. The assay panel consisted of 5 whole organism assays measuring apical effects and 14 cell- and organism-based bioassays with more specific effect observations. Twelve organic water pollutants of diverse structure and unique known modes of action were studied individually and as mixtures mirroring exposure scenarios in freshwaters. We compared the observed mixture effects against component-based mixture effect predictions derived from additivity expectations (assumption of non-interaction). Most of the assays detected the mixture response of the active components as predicted even against a background of other inactive contaminants. When none of the mixture components showed any activity by themselves then the mixture also was without effects. The mixture effects observed using apical endpoints fell in the middle of a prediction window defined by the additivity predictions for concentration addition and independent action, reflecting well the diversity of the anticipated modes of action. In one case, an unexpectedly reduced solubility of one of the mixture components led to mixture responses that fell short of the predictions of both additivity mixture models. The majority of the specific cell- and organism-based endpoints produced mixture responses in agreement with the additivity expectation of concentration addition. Exceptionally, expected (additive) mixture response did not occur due to masking effects such as general toxicity from other compounds. Generally, deviations from an additivity expectation could be explained due to experimental factors, specific limitations of the effect endpoint or masking side effects such as cytotoxicity in in vitro assays. The majority of bioassays were able to quantitatively detect the predicted non-interactive, additive combined effect of the specifically bioactive compounds against a background of complex mixture of other chemicals in the sample. This supports the use of a combination of chemical and bioanalytical monitoring tools for the identification of chemicals that drive a specific mixture effect. Furthermore, we demonstrated that a panel of bioassays can provide a diverse profile of effect responses to a complex contaminated sample. This could be extended towards representing mixture adverse outcome pathways. Our findings support the ongoing development of bioanalytical tools for (i) compiling comprehensive effect-based batteries for water quality assessment, (ii) designing tailored surveillance methods to safeguard specific water uses, and (iii) devising strategies for effect-based diagnosis of complex contamination.