How the Xenopus eleutheroembryonic thyroid assay compares to the amphibian metamorphosis assay for detecting thyroid active chemicals.

The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5'-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA.

Are changes in vitellogenin concentrations in fish reliable indicators of chemical-induced endocrine activity?

Vitellogenin (VTG), a biomarker for endocrine activity, is a mechanistic component of the regulatory assessment of potential endocrine-disrupting properties of chemicals. This review of VTG data is based on changes reported for 106 substances in standard fish species. High intra-study and inter-laboratory variability in VTG concentrations was confirmed, as well as discrepancies in interpretation of results based on large differences between fish in the dilution water versus solvent control, or due to the presence of outlier measurements. VTG responses in fish were ranked against predictions for estrogen receptor agonist activity and aromatase inhibition from bioactivity model output and ToxCast in vitro assay results, respectively. These endocrine mechanisms explained most of the VTG responses in the absence of systemic toxicity, the magnitude of the VTG response being proportional to the in vitro potency. Interpretation of the VTG data was sometimes confounded by an alternative endocrine mechanism of action. There was evidence for both false positive and negative responses for VTG synthesis, but overall, it was rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish in the absence of systemic toxicity. To increase confidence in the VTG results, we recommend improvements in the VTG measurement methodologies and greater transparency in reporting of VTG data (including quality control criteria for assay performance). This review supports the application of New Approach Methodologies (NAMs) by demonstrating that endocrine activity in vitro from mammalian cell lines is predictive for in vivo VTG response in fish, suggesting that in vitro mechanistic data could be used more broadly in decision-making to help reduce animal testing.

An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice.

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines.

Detection of anti-androgenic activity of chemicals in fish studies: a data review.

A systematic review was conducted on the sensitivity of fish testing guidelines to detect the anti-androgenic activity of substances. Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) was used to investigate the conservation of the androgen receptor (AR) between humans and fish, and among fish species recommended in test guidelines. The AR is conserved between fish species and humans (i.e. ligand binding domain [LBD] homology ≥70%) and among the recommended fish species (LBD homology >85%). For model anti-androgens, we evaluated literature data on in vitro anti-androgenic activity in fish-specific receptor-based assays and changes in endpoints indicative of endocrine modulation from in vivo studies. Anti-androgenic activity was most consistently and reliably detected in in vitro and in vivo mechanistic studies with co-exposure to an androgen (spiggin in vitro assay, Rapid Androgen Disruption Activity Reporter [RADAR] Assay, and Androgenised Female Stickleback Screen). Regardless of study design (Fish Short-Term Reproduction Assay [FSTRA], Fish Sexual Development Test [FSDT], partial or full life-cycle tests), or endpoint (vitellogenin, secondary sexual characteristics, gonadal histopathology, sex ratio), there was no consistent evidence for detecting anti-androgenic activity in studies without androgen co-exposure, even for the most potent substances (while less potent substances may induce no (clear) response). Therefore, based on studies without androgen co-exposure (35 FSTRAs and 22 other studies), the other studies (including the FSDT) do not outperform the FSTRA for detecting potent anti-androgenic activity, which if suspected, would be best addressed with a RADAR assay. Overall, fish do not appear particularly sensitive to mammalian anti-androgens.

Harnessing Modeling for Assessing the Population Relevance of Exposure to Endocrine-Active Chemicals.

The presence of endocrine-active chemicals (EACs) in the environment continues to cause concern for wildlife given their potential for adverse effects on organisms. However, there is a significant lack of understanding about the potential effects of EACs on populations. This has real-world limitations for EAC management and regulation, where the aim in environmental risk assessment is to protect populations. We propose a methodological approach for the application of modeling in addressing the population relevance of EAC exposure in fish. We provide a case study with the fungicide prochloraz to illustrate how this approach could be applied. We used two population models, one for brown trout (Salmo trutta; inSTREAM) and the other for three-spined stickleback (Gasterosteus aculeatus) that met regulatory requirements for development and validation. Effects data extracted from the literature were combined with environmentally realistic exposure profiles generated with the FOCUS SW software. Population-level effects for prochloraz were observed in some modeling scenarios (hazard-threshold [HT]) but not others (dose-response), demonstrating the repercussions of making different decisions on implementation of exposure and effects. The population responses, defined through changes in abundance and biomass, of both trout and stickleback exposed to prochloraz were similar, indicating that the use of conservative effects/exposure decisions in model parameterization may be of greater significance in determining population-level adverse effects to EAC exposure than life-history characteristics. Our study supports the use of models as an effective approach to evaluate the adverse effects of EACs on fish populations. In particular, our HT parameterization is proposed for the use of population modeling in a regulatory context in accordance with Commission Regulation (EU) 2018/605. Environ Toxicol Chem 2023;42:1624-1640. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Current testing programs for pesticides adequately capture endocrine activity and adversity for protection of vertebrate wildlife.

The toxicity and ecotoxicity of pesticide active ingredients are evaluated by a number of standardized test methods using vertebrate animals. These standard test methods are required under various regulatory programs for the registration of pesticides. Over the past two decades, additional test methods have been developed with endpoints that are responsive to endocrine activity and subsequent adverse effects. This article examines the available test methods and their endpoints that are relevant to an assessment of endocrine-disrupting properties of pesticides. Furthermore, the article highlights how weight-of-evidence approaches should be applied to determine whether an adverse response in (eco)toxicity tests is caused by an endocrine mechanism of action. The large number of endpoints in the current testing paradigms for pesticides make it unlikely that endocrine activity and adversity is being overlooked. Integr Environ Assess Manag 2023;19:1089-1109. © 2023 Bayer CropScience and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Tube length of chironomid larvae as an indicator for dissolved oxygen in water bodies.

Tube-building larvae of non-biting midges, or chironomids, are considered bioindicators of water pollution. The larvae use benthic particles to make their tubes and create a respiratory current with the movement of their bodies inside the tubes. The tube length of the chironomid larvae varies depending on several physicochemical properties of the aquatic medium. Here we study the role of physicochemical parameters on the tube length from different field sites and in the laboratory. It appears that among different physicochemical factors, dissolved oxygen (DO) has a major role in determining the tube length of the larvae. A quantitative relationship between oxygen concentration and the tube length of larvae is presented here. Our study reveals a longer tube in aquatic media with oxygen deficiency and a shorter tube in those with higher oxygen. This result may help to assess the quality of water bodies and, in particular the status of DO.

Investigating endocrine-disrupting properties of chemicals in fish and amphibians: Opportunities to apply the 3Rs.

Many regulations are beginning to explicitly require investigation of a chemical's endocrine-disrupting properties as a part of the safety assessment process for substances already on or about to be placed on the market. Different jurisdictions are applying distinct approaches. However, all share a common theme requiring testing for endocrine activity and adverse effects, typically involving in vitro and in vivo assays on selected endocrine pathways. For ecotoxicological evaluation, in vivo assays can be performed across various animal species, including mammals, amphibians, and fish. Results indicating activity (i.e., that a test substance may interact with the endocrine system) from in vivo screens usually trigger further higher-tier in vivo assays. Higher-tier assays provide data on adverse effects on relevant endpoints over more extensive parts of the organism's life cycle. Both in vivo screening and higher-tier assays are animal- and resource-intensive and can be technically challenging to conduct. Testing large numbers of chemicals will inevitably result in the use of large numbers of animals, contradicting stipulations set out within many regulatory frameworks that animal studies be conducted as a last resort. Improved strategies are urgently required. In February 2020, the UK's National Centre for the 3Rs and the Health and Environmental Sciences Institute hosted a workshop ("Investigating Endocrine Disrupting Properties in Fish and Amphibians: Opportunities to Apply the 3Rs"). Over 50 delegates attended from North America and Europe, across academia, laboratories, and consultancies, regulatory agencies, and industry. Challenges and opportunities in applying refinement and reduction approaches within the current animal test guidelines were discussed, and utilization of replacement and/or new approach methodologies, including in silico, in vitro, and embryo models, was explored. Efforts and activities needed to enable application of 3Rs approaches in practice were also identified. This article provides an overview of the workshop discussions and sets priority areas for follow-up. Integr Environ Assess Manag 2022;18:442-458. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.

The amphibian metamorphosis assay (AMA; US Environmental Protection Agency [USEPA] test guideline 890.1100 and Organisation for Economic Co-Operation and Development test guideline 231) has been used for more than a decade to assess the potential thyroid-mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the USEPA, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA test guideline, including a modification to a fixed-stage design rather than a fixed-time (i.e., 21-d) design. We describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop-Faber [NF] stage 62), to address both the issues with the fixed-time design and the specific question of thyroid-mediated adversity in a shorter assay than the larval amphibian growth and development assay (LAGDA; Organisation for Economic Co-Operation and Development test guideline 241), using fewer animals and resources. A demonstration study was conducted with the EAMA (up to NF stage 58) using sodium perchlorate. Data analyses and interpretation of the fixed-stage design of the EAMA are more straightforward than the fixed-time design because the fixed-stage design avoids confounded morphometric measurements and thyroid histopathology caused by varying developmental stages at test termination. It also results in greater statistical power to detect metamorphic delays than the fixed-time design. By preferentially extending the AMA to NF stage 62, suitable data can be produced to evaluate thyroid-mediated adversity and preclude the need to perform a LAGDA for thyroid mode of action analysis. The LAGDA remains of further interest should investigations of longer term effects related to sexual development modulated though the hypothalamus-pituitary-gonadal axis be necessary. However, reproduction assessment or life cycle testing is currently not addressed in the LAGDA study design. This is better addressed by higher tier studies in fish, which should then include specific thyroid-related endpoints. Environ Toxicol Chem 2021;40:2135-2144. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Critical Review of Read-Across Potential in Testing for Endocrine-Related Effects in Vertebrate Ecological Receptors.

Recent regulatory testing programs have been designed to evaluate whether a chemical has the potential to interact with the endocrine system and could cause adverse effects. Some endocrine pathways are highly conserved among vertebrates, providing a potential to extrapolate data generated for one vertebrate taxonomic group to others (i.e., biological read-across). To assess the potential for biological read-across, we reviewed tools and approaches that support species extrapolation for fish, amphibians, birds, and reptiles. For each of the estrogen, androgen, thyroid, and steroidogenesis (EATS) pathways, we considered the pathway conservation across species and the responses of endocrine-sensitive endpoints. The available data show a high degree of confidence in the conservation of the hypothalamus-pituitary-gonadal axis between fish and mammals and the hypothalamus-pituitary-thyroid axis between amphibians and mammals. Comparatively, there is less empirical evidence for the conservation of other EATS pathways between other taxonomic groups, but this may be due to limited data. Although more information on sensitive pathways and endpoints would be useful, current developments in the use of molecular target sequencing similarity tools and thoughtful application of the adverse outcome pathway concept show promise for further advancement of read-across approaches for testing EATS pathways in vertebrate ecological receptors. Environ Toxicol Chem 2020;39:739-753. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Does hepatotoxicity interfere with endocrine activity in zebrafish (Danio rerio)?

Vitellogenin (VTG), a well-established biomarker for the diagnosis of endocrine activity in fish, is used in multiple OECD test guidelines (TG) to identify activities of chemicals on hormonal pathways. However, the synthesis of VTG may not only be modified by typical endocrine-related pathways, but also through non-endocrine-mediated processes. In particular, hepatotoxicity, i.e. toxicant-induced impairment of liver structure and function, might influence VTG as a biomarker, since VTG is synthesized in hepatocytes. An intimate understanding of the interplay between endocrine-related and non-endocrine-related pathways influencing VTG production is crucial for the avoidance of erroneous diagnoses in hazard assessment for regulatory purposes of chemical compounds. In order to investigate whether hepatotoxicity may interfere with hepatic VTG synthesis, adult zebrafish (Danio rerio) were exposed to three well-known hepatotoxicants, acetaminophen, isoniazid and acetylsalicylic acid, according to OECD TG 230. Various hepatotoxicity- and endocrine system-related endpoints were recorded: mRNA expression of selected endocrine- and hepatotoxicity-related marker genes in the liver; VTG levels in head/tail homogenates; and liver histopathology. All three test compounds induced significant, but mild single cell necrosis of hepatocytes and transcriptional changes of hepatotoxicity-related marker genes, thus confirming hepatotoxic effects. A positive correlation between hepatotoxicity and reduced hepatic VTG synthesis was not observed, with the single exception of a weak increase in female zebrafish exposed to APAP. This suggests that - in studies conducted according to OECD TG 229 or 230 - it is unlikely that hepatotoxic chemicals will interfere with the hepatic capacity for VTG synthesis.

Interpretation of sexual secondary characteristics (SSCs) in regulatory testing for endocrine activity in fish.

Secondary sexual characteristics (SSCs) are important features that have evolved in many fish species because of inter-individual competition for mates. SSCs are crucial not only for sexual selection, but also for other components of the reproductive process and parental care. Externally, they are especially clear in males (for instance, tubercles, fatpad, anal finnage, colouration) but are also externally present in the females (for instance, ovipositor). These characters are under hormonal control and as such there has been much interest in incorporating them as measures in fish test methods to assess the potential endocrine activity of chemicals. Here we describe the external SSCs in typical laboratory test species for endocrine testing - fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus L.). We also provide some examples and discuss the utility of SSC responses to the endocrine activity of chemicals in the field and the laboratory. This paper is not aimed to provide a comprehensive review of SSCs in fish but presents a view on the assessment of SSCs in regulatory testing. Due to the current regulatory importance of establishing an endocrine mode-of-action for chemicals, we also consider other, non-endocrine factors that may lead to SSC responses in fish. We conclude with recommendations for how the assessment of SSCs in fish could be usefully incorporated into the endocrine hazard and risk assessment of chemicals.

Is normalized hindlimb length measurement in assessment of thyroid disruption in the amphibian metamorphosis assay relevant?

The amphibian metamorphosis assay represents an OECD Level 3 and EDSP Tier 1 ecotoxicity test assessing thyroid activity of chemicals in African clawed frog (Xenopus laevis). To evaluate the effectiveness of snout-vent length (SVL) normalization of hindlimb length (HLL), correlation between the HLL and SVL or body weight was evaluated in the control groups of 10 individual studies from three laboratories. Two studies required separate analysis of the Nieuwkoop-Faber (NF) stage ≤60 and >60 animals creating a total of 12 data sets. On study day 7, significant positive correlation between HLL and SVL or body weight was observed in eight and seven of the 10 data sets, respectively (r = 0.608-0.843 and 0.583-0.876). On study day 21, significant positive correlation between HLL and SVL or body weight was found in three and four of the 12 data sets, respectively (r = 0.452, 0.480 and 0.553 and r = 0.621, 0.546, 0.564 and 0.378). Significant positive correlation between HLL and SVL was found in three of five studies, including ≤NF stage 60 data (r = 0.564, 0.546 and 0.621). In one of eight studies, including >NF stage 60 data, the positive correlation between HLL and body weight was determined (r = 0.378). Negative or no correlation between HLL and SVL or body weight was found in the other late stage data sets. Therefore, use of SVL-normalized HLL to assess thyroid-mediated effects in X. laevis tadpoles is not warranted. HL stage relative to body stage should be considered.

Assessing the population relevance of endocrine-disrupting effects for nontarget vertebrates exposed to plant protection products.

The European Commission intends to protect vertebrate wildlife populations by regulating plant protection product (PPP) active substances that have endocrine-disrupting properties with a hazard-based approach. In this paper we consider how the Commission's hazard-based regulation and accompanying guidance can be operationalized to ensure that a technically robust process is used to distinguish between substances with adverse population-level effects and those for which it can be demonstrated that adverse effects observed (typically in the laboratory) do not translate into adverse effects at the population level. Our approach is to use population models within the adverse outcome pathway framework to link the nonlinear relationship between adverse effects at the individual and population levels in the following way: (1) use specific protection goals for focal wildlife populations within an ecosystem services framework; (2) model the effects of changes in population-related inputs on focal species populations with individual-based population models to determine thresholds between negligible and nonnegligible (i.e., adverse) population-level effects; (3) compare these thresholds with the relevant endpoints from laboratory toxicity tests to determine whether they are likely to be exceeded at hazard-based limits or the maximum tolerated dose/concentration from the experimental studies. If the population threshold is not exceeded, then the substance should not be classified as an endocrine disruptor with population-relevant adversity unless there are other lines of evidence within a weight-of-evidence approach to challenge this. We believe this approach is scientifically robust and still addresses the political and legal requirement for a hazard-based assessment. Integr Environ Assess Manag 2019;15:278-291. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Endocrine Disruption: Current approaches for regulatory testing and assessment of plant protection products are fit for purpose.

The ongoing debate concerning the regulation of endocrine disruptors, has increasingly led to questions concerning the current testing of chemicals and whether this is adequate for the assessment of potential endocrine disrupting effects. This paper describes the current testing approaches for plant protection product (PPP) active substances in the European Union and the United States and how they relate to the assessment of endocrine disrupting properties for human and environmental health. This includes a discussion of whether the current testing approaches cover modalities other than the estrogen, androgen, thyroid and steroidogenesis (EATS) pathways, sensitive windows of exposure, adequate assessment of human endocrine disorders and wildlife species, and the determination of thresholds for endocrine disruption. It is concluded, that the scope and nature of the core and triggered data requirements for PPP active substances are scientifically robust to address adverse effects mediated through endocrine mode(s) of action and to characterise these effects in terms of dose response.