•We characterised the reproductive responses of the fathead minnow to ethinyestradiol and levonorgestrel and a binary mixture of the two.•A new statistical approach was developed to analyse egg ...production during exposure experiments.•The concept of concentration addition was tested.•Plasma drug concentrations and the read across approach were used to guide the interpretation of the reproductive results.
The aquatic environment is polluted with thousands of chemicals. It is currently unclear which of these pose a significant threat to aquatic biota. The typical exposure scenario is now represented by a widespread blanket of contamination composed of myriads of individual pollutants—each typically present at a low concentration. The synthetic steroids, 17α-ethinylestradiol and levonorgestrel, have been widely reported to be present in the aquatic environment in the low ng to sub-ng/l range. They are widely used in contraceptive formulations, both individually and in combination. Our research employed the fathead minnow (Pimephales promelas) 21 day ‘pair-breeding’ assay to assess reproductive output when pairs of fish were exposed to the single chemicals at low environmentally relevant concentrations, and then to a binary mixture of them. A variety of endpoints were assessed, including egg production, which was inhibited in a concentration-dependent manner by both the individual chemicals and the mixture. Significant, sex specific effects were also seen with both chemicals, at differing levels of biological organisation. Plasma concentrations of EE2 and levonorgestrel were predicted and in the case of levonorgestrel measured, and compared with the human therapeutic plasma concentrations (Read-Across approach) to support the interpretation of the results. A novel quantitative method was developed for the data analysis, which ensured a suitable endpoint for the comparative mixture assessment. This approach compares the reproductive performance from individual pairs of fish during chemical exposure to its pre-treatment performance. The responses from the empirical mixture study were compared to predictions derived from the single substance data. We hypothesised combined responses which were best described by the concept of concentration addition, and found no clear indications against this additivity expectation. However, the effect profiles support the current knowledge that both compounds act in different ways to reduce egg production in fish, and suggest that probably response addition (also called Independent action) is the more appropriate mixture model in this case.
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).
Display omitted
•An in vitro testing battery (IVB) that allows screening of chemicals for developmental neurotoxicity (DNT) has been assembled.•Performance estimates (>80% accuracy) have been obtained for the IVB, based on 45 negative/positive controls.•Concentration-response data for altogether 120 compounds have been obtained for ten tests covering altogether 21 endpoints.•Gaps of the IVB have been analyzed, and recommendations for the use of the IVB for regulatory testing have been put forward.
Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis
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
data to anticipate such
effects exist. Prioritization schemes that limit unnecessary
testing are urgently needed.
The aim was to develop a quantitative
to
extrapolation (QIVIVE) approach for predicting
anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats.
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
active concentrations for AR antagonism and androgen synthesis suppression.
We first evaluated our model by using
and
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
evidence for anti-androgenicity but missing
data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid,
-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (
, 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.
Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as
anti-androgens. With the examples investigated, our approach shows great promise for predicting
anti-androgenicity (i.e., AGD shortening) for chemicals with
activity and for minimizing unnecessary
testing. https://doi.org/10.1289/EHP6774.
Evidence is mounting that chemicals can produce joint toxicity even when combined at levels that singly do not pose risks. Environmental Quality Standards (EQS) defined for single pollutants under ...the Water Framework Directive (WFD) do not protect from mixture risks, nor do they enable prioritization of management options. Despite some provisions for mixtures of specific groups of chemicals, the WFD is not fit for purpose for protecting against or managing the effects of coincidental mixtures of water-borne pollutants. The conceptual tools for conducting mixture risk assessment are available and ready for use in regulatory and risk assessment practice. Extension towards impact assessment using cumulative toxic unit and mixture toxic pressure analysis based on chemical monitoring data or modelling has been suggested by the SOLUTIONS project. Problems exist in the availability of the data necessary for mixture risk assessments. Mixture risk assessments cannot be conducted without essential input data about exposures to chemicals and their toxicity. If data are missing, mixture risk assessments will be biassed towards underestimating risks. The WFD itself is not intended to provide toxicity data. Data gaps can only be closed if proper feedback links between the WFD and other EU regulations for industrial chemicals (REACH), pesticides (PPPR), biocides (BPR) and pharmaceuticals are implemented. Changes of the WFD alone cannot meet these requirements. Effect-based monitoring programmes developed by SOLUTIONS should be implemented as they can capture the toxicity of complex mixtures and provide leads for new candidate chemicals that require attention in mixture risk assessment. Efforts of modelling pollutant levels and their anticipated mixture effects in surface water can also generate such leads. New pollutant prioritization schemes conceived by SOLUTIONS, applied in the context of site prioritization, will help to focus mixture risk assessments on those chemicals and sites that make substantial contributions to mixture risks.
The aim of this study was to assess whether the joint effects of three androgen receptor antagonists (vinclozolin, flutamide, procymidone) on male sexual differentiation after in utero and postnatal ...exposures can be predicted based on dose-response data of the individual chemicals.
Test chemicals and mixtures were administered by gavage to time-mated nulliparous, young adult Wistar rats from gestational day 7 to the day before expected birth, and from postnatal days 1-16. Changes in anogenital distance (AGD) and nipple retention (NR) in male offspring rats were chosen as end points for extensive dose-response studies. Vinclozolin, flutamide, and procymidone were combined at a mixture ratio proportional to their individual potencies for causing retention of six nipples in male offspring.
With AGD as the end point, the joint effects of the three anti-androgens were essentially dose additive. The observed responses for NR were slightly higher than those expected on the basis of dose addition. A combination of doses of each chemical, which on its own did not produce statistically significant AGD alterations, induced half-maximal mixture effects. At individual doses associated with only modest effects on NR, the mixture induced NR approaching female values in the males.
Effects of a mixture of similarly acting anti-androgens can be predicted fairly accurately on the basis of the potency of the individual mixture components by using the dose addition concept. Exposure to anti-androgens, which individually appears to exert only small effects, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.
•A combined exposure of fish to pharmaceutical anti-androgens and steroid oestrogens.•Concentrations determined by predictive modelling of environmental scenarios.•The anti-androgens had no ...significant impact on sexual disruption in fish.•A mixture of steroid oestrogens induced intersex at environmental concentrations.•A control treatment unexpectedly contained 100% phenotypic male fish.
Sexual disruption in wild fish has been linked to the contamination of river systems with steroid oestrogens, including the pharmaceutical 17α-ethinylestradiol, originating from domestic wastewaters. As analytical chemistry has advanced, more compounds derived from the human use of pharmaceuticals have been identified in the environment and questions have arisen as to whether these additional pharmaceuticals may also impact sexual disruption in fish. Indeed, pharmaceutical anti-androgens have been shown to induce such effects under laboratory conditions. These are of particular interest since anti-androgenic biological activity has been identified in the aquatic environment and is potentially implicated in sexual disruption alone and in combination with steroid oestrogens. Consequently, predictive modelling was employed to determine the concentrations of two anti-androgenic human pharmaceuticals, bicalutamide and cyproterone acetate, in UK sewage effluents and river catchments and their combined impacts on sexual disruption were then assessed in two fish models. Crucially, fish were also exposed to the anti-androgens in combination with steroid oestrogens to determine whether they had any additional impact on oestrogen induced feminisation. Modelling predicted that the anti-androgenic pharmaceuticals were likely to be widespread in UK river catchments. However, their concentrations were not sufficient to induce significant responses in plasma vitellogenin concentrations, secondary sexual characteristics or gross indices in male fathead minnow or intersex in Japanese medaka alone or in combination with steroid oestrogens. However, environmentally relevant mixtures of oestrone, 17β-oestradiol and 17α-ethinylestradiol did induce vitellogenin and intersex, supporting their role in sexual disruption in wild fish populations. Unexpectedly, a male dominated sex ratio (100% in controls) was induced in medaka and the potential cause and implications are briefly discussed, highlighting the potential of non-chemical modes of action on this endpoint.
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 multiplex
hybridisation (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.
In freshwater systems located in agricultural areas, organisms are exposed to a multitude of toxicologically and structurally different pesticides. For regulatory purposes it is of major importance ...whether the combined hazard of these substances can be predictively assessed from the single substance toxicity. For artificially designed multi-component mixtures, it has been shown that the mixture toxicity can be predicted by
concentration addition (CA) in case of similarly acting substances and by
independent action (IA), if mixtures are composed of dissimilarly acting substances. This study aimed to analyse whether these concepts may also be used to predictively assess the toxicity of environmentally realistic mixtures. For this purpose a mixture of 25 pesticides, which reflects a realistic exposure scenario in field run-off water, was studied for its effects on the reproduction of the freshwater alga
Scenedesmus vacuolatus. The toxicity of the tested mixtures showed a good predictability by CA. This is consistent with the finding that the toxicity was dominated by a group of similarly acting photosystem II inhibitors, although the mixture included substances with diverse and partly unknown mechanisms of action.
IA slightly underestimated the actual mixture toxicity. However, the EC
50 values that can be derived from each prediction, according to CA respectively IA, only differed by a factor of 1.3. The finding of such a small difference is partly explainable by the fact that only few components dominate the mixture scenario in terms of so-called
toxic units (TUs). This connection is established by developing an equation that allows to calculate the maximum possible ratio between corresponding predictions of effect concentrations by IA and CA for any given ratio of the TUs of mixture components, irrespective of their individual concentration–response functions and independent from their mechanisms of action. To evaluate whether small quantitative differences between EC
50 values predicted by CA and IA are an exception or rather the rule for agricultural exposure scenarios, this calculation was applied on an additional set of 18 pesticide exposure scenarios that were taken from the literature. For these scenarios, EC
50 values predicted by IA can never exceed those predicted by CA by more than a factor of 2.5. The findings of this study support the view that CA provides a precautious but not overprotective approach to the predictive hazard assessment of pesticide mixtures under realistic exposure scenarios, irrespective of the similarity or dissimilarity of their mechanisms of action.
In vitro assays for anti-androgens have been developed as screening tools for the identification of androgen receptor (AR) antagonists. We explored the usefulness of such assays for experimental ...purposes that require quantitation of effects in a highly reproducible manner, such as multi-component mixture experiments or evaluation of extracts of complex environmental samples. We have investigated sources of experimental variation in the MDA-kb2 assay for AR-antagonists. By omitting phenol red from culture media, avoiding media changes and extending the period allowed for cell attachment, the dynamic range increased. Variations in luminescence readings decreased, with smaller coefficients of variation within- and between-experiments. Normalisation of luminescence values to positive controls improved experiment-to-experiment reproducibility and allowed pooling of data from independent experiments. We also performed statistical power analyses to determine the minimal suppression of androgenic (DHT) effects by test agents that are detectable as statistically significantly different from positive controls (so-called minimum significant differences, MSD). Using the modified assay protocol extensive concentration–response analyses were conducted with bisphenol A, BDE100 and vinclozolin. Our modified procedure improves considerably the reproducibility of the MDA-kb2 assay.
PDF
