Control performance of the Extended Amphibian Metamorphosis Assay with the African clawed frog Xenopus laevis

The Amphibian Metamorphosis Assay (AMA) was established as a standardized test guideline with the primary purpose of evaluating test materials for potential endocrine activity in the hypothalamus-pituitary-thyroid axis of developing African clawed frogs (Xenopus laevis). The Extended Amphibian Metamorphosis Assay (EAMA) is similar to the AMA with a key difference in that the EAMA terminates at a fixed developmental stage (i.e., Nieuwkoop and Faber [NF] stage 62) rather than at the fixed exposure time of the AMA (i.e., 21 days). This study summarizes the control data at NF stage 62 for a total of 17 EAMAs conducted at two different laboratories. The EAMA termination endpoints, including wet body weight, snout-to-vent length, hind limb length, normalized hind limb length, and thyroid histopathology, as well as the time to reach NF stage 62, are summarized. The endpoints in the EAMA exhibited less variability in comparison to endpoints collected at termination (Day 21) in the AMA. Regression analyses were conducted for continuous endpoints taken at the termination of the EAMA. Wet body weight versus snout-to-vent length showed the highest mean coefficient of determination of R2 = 0.71. Time to NF stage 62 did not show a strong correlation to morphometric endpoints (R2 ≤ 0.40), which may reflect the plasticity of amphibian metamorphosis. Histopathological investigations of thyroid glands indicated that mild and moderate follicular cell hypertrophy and mild follicular cell hyperplasia occur regularly as a part of normal development among NF stage 62 control tadpoles. In summary, the control performance data of 17 EAMAs indicate a robust baseline that can be used to refine the performance criteria and aid in the interpretation of the results of future fixed-stage test designs.