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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]

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]