Interlaboratory validation of the human thyroid microtissue assay.

The U.S. Environmental Protection Agency (EPA) continues to evaluate strategies to implement new approach methods (NAMs) for screening chemicals that disrupt the thyroid endocrine system. Validation of NAMs is a critical milestone toward establishing confidence in data sources that could be used in a regulatory decision-making context. The objective of this study was to conduct an interlaboratory validation of the human thyroid microtissue assay to evaluate its relevance and reliability. In coordination with the US validation authority, NICEATM, and collaboration with industry partners (LifeNet Health, Bayer Crop Science, Corteva Agrisciences), the study aims were to (1) define the study design and establish standard operating procedures, (2) conduct test method transfer, training, and within-laboratory model performance evaluation, (3) perform interlaboratory reference chemical testing and assay performance evaluation. Progress was independently monitored by a validation management team comprised of an international group of experts in thyroid physiology, in vitro test methods, and regulatory toxicology. Results indicated the thyroid microtissue model could be reliably transferred to new laboratories with reproducible effects on thyroid hormone synthesis. Interlaboratory testing of 4 blinded reference chemicals (3 true positive, 1 true negative) across 3 independent human donors revealed consistent bioactivity across the reference set and performance metrics (dynamic range, precision, screening quality) that met acceptance criteria and shed insight into areas for improvement. In the context of a NAM-based testing strategy, a validated human thyroid microtissue assay enables direct measurement of thyroid hormone synthesis perturbations, reducing reliance on animal testing and addressing a critical mode-of-action that is of regulatory concern.