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Reference chemicals were selected based on thyroid bioactivity in 'Tier 1' screening assays used by the U.S. EPA's Endocrine Disruptor Screening Program. Active reference chemicals had significant effects on thyroid-responsive endpoints in the amphibian metamorphosis assay, and the male and female pubertal rat assays. In the absence of thyroid weight or histopathological effects, additional published studies providing mechanistic data on thyroid activity were required for active chemicals. Inactive reference chemicals had no significant effects on thyroid-responsive endpoints in Tier 1 assays, or in amphibian or rodent studies from several online databases. The 34 reference chemicals (29 active and five inactive) will be useful for performance-based validation of alternative, high throughput screening assays for thyroid bioactivity. (C) 2016 Published by Elsevier Inc.