Marini JP et al. (2023), An Amphibian Metamorphosis Assay Dietary Restri...

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Environ Toxicol Chem 2023 May 01;425:1061-1074. doi: 10.1002/etc.5596.

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

Marini JP , Wolf JC , Mingo V , Sayers LE , Jamieson SY , Wheeler JR .

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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.

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Species referenced: Xenopus laevis
GO keywords: endocrine process

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	Figure 1 Summarized biological endpoint data for all surviving tadpoles evaluated at 21 days. (A) Snout–vent length (SVL), (B) hindlimb length (HLL), (C) normalized hindlimb length (nHLL), (D) body weight, (F) developmental stage. The minimum and maximum values for each endpoint are represented by the bottom and top whiskers of the plot (lowest and highest data points in the data set excluding outliers, respectively). The lower quartile (25th percentile) is represented by the bottom of the box, and the upper quartile (75th percentile) is represented by the top. The continuous line in the middle of the plot represents the endpoint median. ° = outliers; * = statistically significant reduction compared with the 50% ration (one-tailed [C > thyroid] Jonckheere–Terpstra step-down test, p < 0.05); ** = statistically significant reduction compared with the 50% ration (one-tailed [C > thyroid] Jonckheere–Terpstra step-down test, p < 0.001). TG = test guideline; NF = Nieuwkoop–Faber.
	Figure 2 Developmental stage distribution at day 21 in the definitive experiment. NF = Nieuwkoop–Faber.
	Figure 3 Thyroid glands from feed-restricted frogs, according to percentage fed. (A and B) 50%, moderate follicular cell (FC) hyperplasia and moderate FC hypertrophy. (C and D) 30%, mild FC hyperplasia and mild FC hypertrophy. (E and F) 20%, mild FC hypertrophy. (G and H) 10%, no remarkable findings. (I and J) 5%, moderate thyroid atrophy. A, C, E, G, and I: scale bar = 100 µm. B, D, F, H, and J: scale bar = 25 µm.
	Figure 4 Livers from feed-restricted frogs, according to percentage fed. (A) 50% male, moderate vacuolation. (B) 50% female, moderate vacuolation. (C) 30% female, mild vacuolation. (D) 20% male, minimal vacuolation. (E) 10% sex undetermined, no vacuolation (scored as not remarkable). (F) 5% sex undetermined, minimal liver atrophy. All images: scale bar = 25 µm.
	Figure 5 Gonads from feed-restricted frogs, according to percentage fed. (A) 50% testis (arrows). (B) 50% ovary (arrows). (C) 5% undifferentiated gonad (arrows). All images: scale bar = 50 µm.