The Extended Amphibian Metamorphosis Assay: A Thyroid‐Specific and Less Animal‐Intensive Alternative to the Larval Amphibian Growth and Development Assay

Abstract

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 fixedstage 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.

Normal Table of Xenopus development: a new graphical resource

“…Similarly, we predict this work will aid the staging of embryos for phenotype scoring for EAMA and AMA (Extended/Amphibian Metamorphosis Assay). EAMA and AMA assess the effects of potential endocrine and thyroid pathway disrupting substances on amphibian metamorphosis, by scoring limb phenotypes, abnormal behavior, time to metamorphosis, and mortality during prometamorphosis and climax metamorphosis stages ( Dang, 2019 ; Ortego et al, 2021 ).…”

Importance of diet in amphibian metamorphosis‐based studies designed to assess the risk of thyroid active substances

“…Kupferberg (1997) highlighted the essential role of algal proteins in growth rate, metamorphosis, and thyroid function in amphibians. Historical studies such as Goleman et al (2002, 2006), DuPreez et al (2008), and more recently Ortego et al (2021) used FB (Nasco), which is a diet formulated for juvenile and adult Xenopus , although it was long marketed as larval food. Current USEPA and OECD guidelines for the AMA recommend the use of Sera Micron (now called Sera Micron Nature [SMN], 2022), which is an algal‐marine protein diet (OECD, USEPA, 2009).…”

Statement concerning the testing strategy and timelines proposed by the applicant for the assessment of the endocrine disruption properties of acibenzolar‐S‐methyl in the context of the review of the approval of the active substance

“…Although it is acknowledged that the validation of the Larval Amphibian Growth and Development Assay (LAGDA) was limited to few chemicals (only one substance known to act through multiple ED MoA including a T‐MoA), it is noted that the extended AMA suggested by Ortego et al. ( 2021 ) is a non‐standardised protocol as well, when comparing it to the existing AMA. In both cases the reproducibility of the results by different laboratories is a source of uncertainty.…”

“…The proposal to deviate from the testing strategy for non‐mammalian species for the T‐modality (i.e. extended AMA according to Ortego et al., 2021 instead of a LAGDA according to OECD TG 241) as proposed in the ECHA/EFSA ED guidance, in case of positive evidence for endocrine activity, as indicated in the interim results of the available AMA, does not seem fully justified. A comparison of the available test methods in terms of, e.g.…”

“…As a consequence, the extended AMA foresees the investigation of time to reach NF stage 62, while in the standard AMA, the developmental stage after 21 days is measured. This measured parameter in the extended AMA may allow employing alternative statistical tools (USEPA, 2013a , b ; Haselman et al., 2016 ; Ortego et al., 2021 ) which might be increasingly informative or powerful. The animals are exposed from stage NF 51 to 57 (21 days) in the case of the AMA, and from stage NF 51 to stage NF 62 (until 95% of the surviving tadpoles reached NF stage 62 or up to 45 days post‐fertilisation, whichever occurred first) in the extended AMA.…”