An Amphibian Metamorphosis Assay Dietary Restriction Study: Lessons for Data Interpretation.

The Amphibian Metamorphosis Assay (AMA) is a key in vivo endocrine screen to investigate potential thyroid active chemicals. The Test Guidelines and associated guidance consider treatment-related effects on thyroid gland histomorphology to 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 5 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. Data derived from this study indicate that treatment-related histopathological changes in the AMA can be induced by non-chemical factors; therefore, histopathologic 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 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, in order to conclude that a test substance has thyroid endocrine activity. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2023;00:0-0. © 2023 SETAC.