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.

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.

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.

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.

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.

A review of the evidence for endocrine disrupting effects of current-use chemicals on wildlife populations.

This review critically examines the data on claimed endocrine-mediated adverse effects of chemicals on wildlife populations. It focuses on the effects of current-use chemicals, and compares their apparent scale and severity with those of legacy chemicals which have been withdrawn from sale or use, although they may still be present in the environment. The review concludes that the effects on wildlife of many legacy chemicals with endocrine activity are generally greater than those caused by current-use chemicals, with the exception of ethinylestradiol and other estrogens found in sewage effluents, which are causing widespread effects on fish populations. It is considered that current chemical testing regimes and risk assessment procedures, at least those to which pesticides and biocides are subjected, are in part responsible for this improvement. This is noteworthy as most ecotoxicological testing for regulatory purposes is currently focused on characterizing apical adverse effect endpoints rather than identifying the mechanism(s) responsible for any observed effects. Furthermore, a suite of internationally standardized ecotoxicity tests sensitive for potential endocrine-mediated effects is now in place, or under development, which should ensure further characterization of substances with these properties so that they can be adequately regulated.

Weight of evidence approaches for the identification of endocrine disrupting properties of chemicals: Review and recommendations for EU regulatory application.

A Weight-of-evidence (WoE) evaluation should be applied in assessing all the available data for the identification of endocrine disrupting (ED) properties of chemicals. The European Commission draft acts specifying criteria under the biocidal products and plant protection products regulations require that WoE is implemented for the assessment of such products. However, only some general considerations and principles of how a WoE should be conducted are provided. This paper reviews WoE approaches to distil key recommendations specifically for the evaluation of potential ED properties of chemicals. In a manner, which is consistent with existing, published WoE frameworks, the WoE evaluation of ED properties can be divided into four phases: 1) Definition of causal questions and data gathering and selection, 2) Review of individual studies, 3) Data integration and evaluation, and 4) Drawing conclusions based on inferences. Recommendations are made on how to conduct each phase robustly and transparently to help guide the WoE evaluation of potential endocrine disrupting properties of chemicals within a European regulatory context.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

The utility of QSARs in predicting acute fish toxicity of pesticide metabolites: A retrospective validation approach.

The European Plant Protection Products Regulation 1107/2009 requires that registrants establish whether pesticide metabolites pose a risk to the environment. Fish acute toxicity assessments may be carried out to this end. Considering the total number of pesticide (re-) registrations, the number of metabolites can be considerable, and therefore this testing could use many vertebrates. EFSA's recent "Guidance on tiered risk assessment for plant protection products for aquatic organisms in edge-of-field surface waters" outlines opportunities to apply non-testing methods, such as Quantitative Structure Activity Relationship (QSAR) models. However, a scientific evidence base is necessary to support the use of QSARs in predicting acute fish toxicity of pesticide metabolites. Widespread application and subsequent regulatory acceptance of such an approach would reduce the numbers of animals used. The work presented here intends to provide this evidence base, by means of retrospective data analysis. Experimental fish LC50 values for 150 metabolites were extracted from the Pesticide Properties Database (http://sitem.herts.ac.uk/aeru/ppdb/en/atoz.htm). QSAR calculations were performed to predict fish acute toxicity values for these metabolites using the US EPA's ECOSAR software. The most conservative predicted LC50 values generated by ECOSAR were compared with experimental LC50 values. There was a significant correlation between predicted and experimental fish LC50 values (Spearman rs = 0.6304, p < 0.0001). For 62% of metabolites assessed, the QSAR predicted values are equal to or lower than their respective experimental values. Refined analysis, taking into account data quality and experimental variation considerations increases the proportion of sufficiently predictive estimates to 91%. For eight of the nine outliers, there are plausible explanation(s) for the disparity between measured and predicted LC50 values. Following detailed consideration of the robustness of this non-testing approach, it can be concluded there is a strong data driven rationale for the applicability of QSAR models in the metabolite assessment scheme recommended by EFSA. As such there is value in further refining this approach, to improve the method and enable its future incorporation into regulatory guidance and practice.

Acute oral toxicity of chemicals in terrestrial life stages of amphibians: Comparisons to birds and mammals.

Amphibians are currently the most threatened and rapidly declining group of vertebrates and this has raised concerns about their potential sensitivity and exposure to plant protection products and other chemicals. Current environmental risk assessment procedures rely on surrogate species (e.g. fish and birds) to cover the risk to aquatic and terrestrial life stages of amphibians, respectively. Whilst a recent meta-analysis has shown that in most cases amphibian aquatic life stages are less sensitive to chemicals than fish, little research has been conducted on the comparative sensitivity of terrestrial amphibian life stages. Therefore, in this paper we address the questions "What is the relative sensitivity of terrestrial amphibian life stages to acute chemical oral exposure when compared with mammals and birds?" and "Are there correlations between oral toxicity data for amphibians and data for mammals or birds?" Identifying a relationship between these data may help to avoid additional vertebrate testing. Acute oral amphibian toxicity data collected from the scientific literature and ecotoxicological databases were compared with toxicity data for mammals and birds. Toxicity data for terrestrial amphibian life stages are generally sparse, as noted in previous reviews. Single-dose oral toxicity data for terrestrial amphibian life stages were available for 26 chemicals and these were positively correlated with LD50 values for mammals, while no correlation was found for birds. Further, the data suggest that oral toxicity to terrestrial amphibian life stages is similar to or lower than that for mammals and birds, with a few exceptions. Thus, mammals or birds are considered adequate toxicity surrogates for use in the assessment of the oral exposure route in amphibians. However, there is a need for further data on a wider range of chemicals to explore the wider applicability of the current analyses and recommendations.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

A review of the effects of azole compounds in fish and their possible involvement in masculinization of wild fish populations.

Endocrine-mediated effects in fish populations have been widely documented. Most attention has been focused on feminization caused by estrogenic substances, but this paper reviews evidence for the effects of a group of fungicides and pharmaceuticals, the azoles, which have been reported to cause masculinization in fish. The paper considers information from laboratory studies on the effects of azole compounds on fish endocrinology, and on the potential existence of such effects in wild fish. The occurrence of some azoles in effluents and surface waters has also been briefly reviewed. Under laboratory conditions, many azoles are able to cause masculinization or defeminization in fish by inhibition of the P450 enzyme aromatase (CYP19). However, in no case where such effects have been observed in the field has a link been established with this group of substances. In most instances, other more convincing explanations have been proposed. Peak concentrations of some azoles in surface waters can approach those which, under continuous long-term exposure in the laboratory, might lead to some aromatase inhibition. However, available data on exposure and effects provide reassurance that the concentrations of azoles found in surface waters are too low to cause adverse effects in fish by interference with their endocrine system. Compared to the widespread observations of feminization and estrogenic effects in (male) fish, there are relatively few papers describing masculinization or defeminization in (female) wild fish populations, suggesting that this is quite a rare phenomenon. The significance of this result is emphasized by the fact that fish are among the best studied organisms in the environment.

Mind the gap: Concerns using endpoints from endocrine screening assays in risk assessment.

Endocrine screening assays not only provide mechanistic information on the potential of a substance to interact with the endocrine system, but also data potentially relevant for risk assessment. However, these screening assays have a number of limitations that should be considered before the direct use of such data for risk assessment purposes. This paper discusses the limitations that should be considered for both human and environmental risk assessment. A proposal is made to provide an objective and transparent process in order to consider which endpoint(s) might be incorporated into a risk assessment, and when more definitive studies may be of value. The proposal is complemented with an easy-to-follow flowchart to aid industry scientists and regulators when evaluating the relevance of these data. Such an approach is necessary to ensure the appropriate use of screening data to further our understanding of the eco/toxicological profile of substances undergoing screening.

Reducing the number of fish in bioconcentration studies with general chemicals by reducing the number of test concentrations.

Fish bioconcentration test guidelines generally require that bioconcentration factors (BCFs) are determined at two exposure concentrations. However, recent revisions to the OECD test guideline for bioconcentration testing (TG 305) provide the option to use only one exposure concentration, when justification is provided, although two concentrations may still be required for some regulatory purposes. Recently, this justification has been demonstrated for plant protection product active ingredients. To determine whether this justification has a broader validity for general chemicals, an analysis of 236 BCF studies on general chemicals was conducted. The results presented here again demonstrate that BCF values do not significantly differ between concentrations when more than one concentration is used. This relationship is particularly strong for BCFs ⩾1000L/kg, which is beneficial, since only chemicals with BCFs >2000L/kg may require regulatory action. This analysis therefore provides a data-driven rationale for using the one test concentration approach for general chemical substances and thus could contribute to a substantial reduction in the use of fish in bioconcentration tests.

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda).

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608µgCdL(-1)). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20±1°C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56days were reproducible between laboratories (68

Occurrence of common frog (Rana temporaria) and common toad (Bufo bufo) adults and metamorphs in agricultural fields in Germany: Potential for exposure to plant protection products.

To characterize the potential for exposure of amphibian terrestrial life stages to plant protection products (PPP), we studied the occurrence and habitat use of adult and metamorph common frogs (Rana temporaria) and common toads (Bufo bufo) in an agricultural landscape in Germany. The four selected study sites were breeding ponds with approximately 80% agricultural land within a 1-km radius. Adults were monitored by radio tracking for two years, and metamorph numbers were assessed for one summer using pitfall traps alongside drift fences. The results demonstrate that adults were rarely present in arable fields at any of the sites (overall 0.5% and 4% of total observations for frogs and toads, respectively). Metamorph captures in arable fields were more variable, ranging from 1.2% to 38.8% (frogs) and from 0.0% to 26.1% (toads) across study sites. Unsurprisingly, most captures in arable fields for both toad and frog metamorphs occurred at the site where the pond was completely surrounded by arable fields. Overall, the presence of adult amphibians in arable fields was limited and, for the metamorphs, occurred primarily when crops were denser and PPP spray interception higher. Diurnal hiding behavior was observed with the highest activity recorded at night, further reducing the risk of dermal exposure from direct PPP overspraying. In addition, it appeared that alternative habitats, such as woody structures or water bodies in the broader surrounding area, were preferred by the animals over the arable areas. The use of buffer zones around water bodies in agricultural areas would be an effective risk mitigation measure to protect terrestrial adults and metamorphs residing there and would reduce spray drift entry into water bodies during PPP application. It is hoped that these results will contribute to the discussion of risk assessment and mitigation options for amphibians. Integr Environ Assess Manag 2024;20:2218-2230. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Endocrine disruption assessment in aquatic vertebrates - Identification of substance-induced thyroid-mediated effect patterns.

According to the World Health Organisation and European Commission definitions, substances shall be considered as having endocrine disrupting properties if they show adverse effects, have endocrine activity and the adverse effects are a consequence of the endocrine activity (using a weight-of-evidence approach based on biological plausibility), unless the adverse effects are not relevant to humans or non-target organisms at the (sub)population level. To date, there is no decision logic on how to establish endocrine disruption via the thyroid modality in non-mammalian vertebrates. This paper describes an evidence-based decision logic compliant with the integrated approach to testing and assessment (IATA) concept, to identify thyroid-mediated effect patterns in aquatic vertebrates using amphibians as relevant models for thyroid disruption assessment. The decision logic includes existing test guidelines and methods and proposes detailed considerations on how to select relevant assays and interpret the findings. If the mammalian dataset used as the starting point indicates no thyroid concern, the Xenopus Eleutheroembryonic Thyroid Assay allows checking out thyroid-mediated activity in non-mammalian vertebrates, whereas the Amphibian Metamorphosis Assay or its extended, fixed termination stage variant inform on both thyroid-mediated activity and potentially population-relevant adversity. In evaluating findings, the response patterns of all assay endpoints are considered, including the direction of changes. Thyroid-mediated effect patterns identified at the individual level in the amphibian tests are followed by mode-of-action and population relevance assessments. Finally, all data are considered in an overarching weight-of-evidence evaluation. The logic has been designed generically and can be adapted, e.g. to accommodate fish tests once available for thyroid disruption assessments. It also ensures that all scientifically relevant information is considered, and that animal testing is minimised. The proposed decision logic can be included in regulatory assessments to facilitate the conclusion on whether substances meet the endocrine disruptor definition for the thyroid modality in non-mammalian vertebrates.

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).