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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]

Abstract: African clawed frogs (Xenopus laevis) were exposed to one of eight nominal waterborne concentrations including 0, 0.1, 1.0, 10, or 25μg/L atrazine, 0.005% ethanol (EtOH), or 0.1mg/L estradiol (E2) or dihydrotestosterone (DHT) containing 0.005% EtOH. Frogs were exposed from 72h posthatch until 2–3 months postmetamorphosis via a 3-day static renewal exposure regimen. Atrazine at concentrations between 0.1 and 25μg/L did not significantly affect mortality, growth, gonad development, laryngeal muscle size, or aromatase activity in juvenile X. laevis. Male frogs exposed to 1.0μg/L atrazine had lower E2 levels compared to controls, but this response was not consistent among other concentrations of atrazine. Male and female frogs exposed to DHT had larger laryngeal dilator muscle areas compared to controls. E2-exposed female frogs had decreased gonadal aromatase activity, and E2-exposed male frogs had statistically greater plasma concentrations of E2 compared to controls. [Copyright &y& Elsevier]

Embryos of the green frog ( Rana clamitans ) were collected from the field and exposed to 1 of 6 water-borne treatments for 273 d (mid July 2001 to mid April 2002). The treatments were 0, 10, or 25 μg/L atrazine, 0.005% ethanol (EtOH), or 0.1 mg/L estradiol or dihydrotestosterone carried in 0.005% EtOH. Treatments were applied in a static renewal system with a 50% test solution replacement approximately every 3 d. Following the exposure period, tadpoles were reared in freshwater until metamorphosis or until study termination (at d 506). Time to initiate and complete metamorphosis, stage-specific mortality, length and weight at metamorphosis, and gross morphology and histology of the gonads were examined. At environmentally relevant concentrations, atrazine did not consistently affect growth or metamorphosis. Compared to controls, the length of the larval period was greater in tadpoles exposed to 10 μg/L atrazine. However, the length of the larval period was not markedly different between tadpoles in the control and 25 μg/L atrazine treatments. Neither gross gonadal morphology nor histopathology of the gonads in postmetamorphic frogs was significantly altered in response to atrazine exposure. This study provides evidence that environmentally relevant concentrations of atrazine do not adversely affect the growth or reproductive development of R. clamitans. [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]