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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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The relative sensitivity of amphibians to chemicals in the environment, including plant protection product active substances, is the subject of ongoing scientific debate. The objective of this study was to compare systematically the relative sensitivity of amphibians and fish to chemicals. Acute and chronic toxicity data were obtained from the U.S. Environmental Protection Agency (U.S. EPA) ECOTOX database and were supplemented with data from the scientific and regulatory literature. The overall outcome is that fish and amphibian toxicity data are highly correlated and that fish are more sensitive (both acute and chronic) than amphibians. In terms of acute sensitivity, amphibians were between 10- and 100-fold more sensitive than fish for only four of 55 chemicals and more than 100-fold more sensitive for only two chemicals. However, a detailed inspection of these cases showed a similar acute sensitivity of fish and amphibians. Chronic toxicity data for fish were available for 52 chemicals. Amphibians were between 10- and 100-fold more sensitive than fish for only two substances (carbaryl and dexamethasone) and greater than 100-fold more sensitive for only a single chemical (sodium perchlorate). The comparison for carbaryl was subsequently determined to be unreliable and that for sodium perchlorate is a potential artifact of the exposure medium. Only a substance such as dexamethasone, which interferes with a specific aspect of amphibian metamorphosis, might not be detected using fish tests. However, several other compounds known to influence amphibian metamorphosis were included in the analysis, and these did not affect amphibians disproportionately. These analyses suggest that additional amphibian testing is not necessary during chemical risk assessment., (Copyright © 2013 SETAC.)

The amphibian metamorphosis assay (AMA) is a key in vivo endocrine screen to investigate chemicals with potential thyroid activity. The test guidelines and associated guidance consider that treatment‐related effects on thyroid gland histomorphology automatically result in the assay being considered positive for thyroid activity, independent of the direction of change or conflicting results in the other biological endpoints. An AMA study was conducted with five different feeding rations equivalent to 50%, 30%, 20%, 10%, and 5% of the recommended feeding rate. Biological endpoints relating to growth and development, including thyroid gland histopathology, were evaluated, and the specificity of these endpoints for the determination of thyroid activity was assessed. There was no effect on survival or clinical signs of toxicity. Effects related to feed reduction generally occurred in a feeding ration–response manner and included reduced development stage; reduced body weight and body length metrics; decreased prevalence of thyroid follicular cell hyperplasia and hypertrophy, and the occurrence of thyroid atrophy; reduced liver vacuolation; and the occurrence of liver atrophy. The results indicate that treatment‐related histopathological changes in the AMA can be induced by Non‐chemical factors; therefore histopathological results are not necessarily diagnostically specific for chemically induced thyroid endocrine activity. Consequently, the interpretation of data from AMA studies should be adjusted accordingly. We recommend that the decision logic presented in the test guidelines and associated guidance be changed to reflect a requirement for directional agreement between the thyroid histopathology findings and the growth and developmental endpoints before it is concluded that a test substance has thyroid endocrine activity. Environ Toxicol Chem 2023;42:1061–1074. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. [ABSTRACT FROM AUTHOR]

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource‐intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward an "animal‐free" testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross‐sector initiative, our review focuses on the current state of the science regarding the use of NAMs to identify chemical‐induced endocrine effects. The present study highlights the challenges of using NAMs for safety assessment and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. New approach methodologies can be integrated as part of a weight‐of‐evidence approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit‐for‐purpose methods to identify chemicals acting via endocrine mechanisms. Environ Toxicol Chem 2023;42:757–777. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. [ABSTRACT FROM AUTHOR]

Amphibians are currently considered to be covered by pesticide Environmental Risk Assessment schemes by surrogacy assumptions of exposure and susceptibility based on typical laboratory test species such as fish, mammals, and birds. While multiple reviews have shown for this approach to be adequate in the case of aquatic stages, the same cannot be definitively stated for terrestrial stages. Concerns have risen that exposure of amphibians is likely to be highly influenced by dermal absorption, primarily due to the high permeability of their skin and the lack of a protective layer, such as fur or feathers. It is thus hypothesized that dermal uptake could be a significant route of exposure. Consequently, it is necessary to determine the relative importance of different exposure routes that might affect the integrated toxicity outcome for terrestrial amphibian life-stages. Here, a one-compartment Toxicokinetic model was derived and tested using a publicly available dataset containing relevant exposure and uptake information for juvenile anurans exposed to 13 different pesticides. Modelled body burdens were then compared to measured burdens for a total of 815 individuals. Overall, a good concordance between modelled and measured values was observed, with the predicted and measured body burdens differing by a factor of 2 on average (overall R2 of 0.80 and correlation coefficient of 0.89), suggesting good predictivity of the model. Accordingly, the model predicts realistic body burdens for a variety of frog and toad species, and overall, for anurans. As the model includes rehydration (implicit in the evaluated studies) but currently does not account for metabolism, it can be seen as a worst-case assessment. We suggest toxicokinetic models, such as the one here presented, could be used to characterize dermal exposure in amphibians, screen for pesticides of concern, and prioritize risk assessment efforts, whilst reducing the need for de novo vertebrate testing. [Display omitted] • A one compartment Toxicokinetic (TK) model is described for anurans. • The model allows to extrapolate body burdens after exposure to contaminated soil. • On average, model predictions deviated from measured data by a factor of 2. • Only in 1% of cases, estimates underestimated exposure by > 10x compared to measured data. • The model could be used to reduce the need de novo vertebrate testing. [ABSTRACT FROM AUTHOR]

The Amphibian Metamorphosis Assay (AMA) is used to identify substances that potentially interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. Although numerous AMA studies have been performed since the establishment of this assay a decade earlier, a comprehensive, large-scale examination of histopathology data obtained from control larvae has not been performed. The current investigation reviewed 51 AMA experiments conducted at 7 different laboratories in Europe and North America. Dilution water control and/or solvent control specimens from each study (1,335 animals total) had been evaluated microscopically by one of eight anatomic pathologists. In order of descending frequency, the most common findings in prometamorphic Xenopus laevis controls were the core criteria of follicular cell (FC) hypertrophy, FC hyperplasia, thyroid hypertrophy, and thyroid atrophy, respectively. Less frequently recorded were non-core and ad hoc diagnoses, the toxicological relevance and utility of which were in some cases uncertain. As anticipated, the prevalence of FC hypertrophy and FC hyperplasia diagnoses were at least partially dependent on the Nieuwkoop and Faber (NF) stage at sacrifice. The recorded frequencies of each of the four core diagnoses also differed according to pathologist, which suggests that pathologist diagnostic interpretation is a potential source of variability across AMA study outcomes. Based on the current examination of the AMA historical data, and further hands-on experience with this assay, diagnostic approaches to evaluating the histopathology endpoint are discussed, and several recommendations are proposed for the refinement of core diagnostic criteria assessment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • We propose a decision logic to identify thyroid disruption in aquatic vertebrates. • The decision logic is evidence-based and includes internationally agreed guidelines. • Thyroid-mediated effect patterns must consider consistency and directions of changes. • The weight-of-evidence considers mode-of-action and population relevance assessments. • The decision logic supports regulatory assessments while minimising animal testing. 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. [ABSTRACT FROM AUTHOR]

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. • The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently developed to screen for thyroid activity of chemicals. • XETA results were compared to Amphibian Metamorphosis Assay (AMA) outcomes for the detection of thyroid-active chemicals. • Results were concordant for all selected mechanisms, but the XETA did not detect inhibition of the sodium-iodide symporter. • Thyroperoxidase inhibition requires further mechanistic investigations due to equivocal responses in the XETA. • The XETA and the AMA consistently identified uridine 5′-diphospho-glucuronosyltransferase inducers as thyroid-inactive. [ABSTRACT FROM AUTHOR]

• The Amphibian Metamorphosis Assay (AMA) and Fish Short Term Reproduction Assay (FSTRA) are standardized regulatory tests used to determine if a chemical might affect the hypothalamic-pituitary-thyroid (HPT) axis or hypothalamic-pituitary-gonadal (HPG) axis, respectively. • Histopathology historical control data were assembled and evaluated from 55 AMA and 45 fathead minnow FSTRA studies. • Historical control data were found useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, differentiating endocrine from non-endocrine effects, identifying background lesions, and helping to determine if specific improvements to established test guidance may be warranted. The Amphibian Metamorphosis Assay (AMA) is used to determine if a tested chemical has potential to impact the hypothalamic-pituitary-thyroid (HPT) axis of Xenopus laevis tadpoles, while the Fish Short Term Reproduction Assay (FSTRA) assesses potential effects on the hypothalamic-pituitary-gonadal (HPG) axis of fish such as the fathead minnow (Pimephales promelas). Several global regulatory programs routinely require these internationally validated tests be performed to determine the potential endocrine activity of chemicals. As such, they are conducted in accordance with standardized protocols and test criteria, which were originally developed more than a decade ago. Sizeable numbers of AMA and FSTRA studies have since been carried out, which allows for the mining of extensive historical control data (HCD). Such data are useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, discriminating target from non-target effects, and for refining current testing paradigms. The present paper provides histopathology HCD from 55 AMA studies and 45 fathead minnow FSTRA studies, so that these data may become publicly available and thus aid in the interpretation of future study outcomes. Histopathology is a key endpoint in these assays, in which it is considered to be one of the most sensitive indicators of endocrine perturbation. In the current review, granular explorations of HCD data were used to identify background lesions, to assess the utility of particular diagnostic findings for distinguishing endocrine from non-endocrine effects, and to help determine if specific improvements to established regulatory guidance may be warranted. Knowledge gleaned from this investigation, supplemented by information from other recent studies, provided further context for the interpretation of AMA and FSTRA histopathology results. We recommend HCDs for the AMA and FSTRA be maintained to support the interpretation of study results. [ABSTRACT FROM AUTHOR]