The Extended Amphibian Metamorphosis Assay (EAMA): A thyroid-specific and less animal-intensive alternative to the Larval Amphibian Growth and Development Assay (LAGDA, OECD TG 241).

The Amphibian Metamorphosis Assay (AMA, OPPTS 890.1100, 2009 and OECD TG 231, 2009) has been used for over a decade to assess potential thyroid (T)-mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the United States Environmental Protection Agency, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA Test Guideline (TG), including a modification to a fixed-stage design rather than a fixed-time (i.e., 21-day) design. In this paper, we describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop-Faber, NF stage 62) in order to address both the issues with the fixed-time design and the specific question of T-mediated adversity in a shorter assay than the Larval Amphibian Growth and Development Assay (LAGDA, OECD TG 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 it 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 T-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 (HPG) 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 T-related endpoints. This article is protected by copyright. All rights reserved.