ArticleNeurotoxicology2026
Zebrafish behavioral assessment as a first-tier whole-organism NAM for developmental neurotoxicity: A multi-laboratory evaluation.
Article in Neurotoxicology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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13 authors.
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Abstract
The development of New Approach Methodologies (NAMs) for developmental neurotoxicity (DNT) assessment requires demonstrating reproducibility across testing facilities and clearly defining their role in tiered testing strategies. This multi-laboratory study evaluated the zebrafish Light-Dark Transition Test (LDTT) across four independent laboratories testing 33 compounds (28 suspected DNT toxicants and 5 putative DNT-negatives), including organophosphate and pyrethroid insecticides (deltamethrin, parathion), flame retardants (TDCIPP, TMPP, BPDP), and neonicotinoids (nicotine, acetamiprid) using agreed-upon protocol elements. A novel three-tiered quality control framework removed 10.9% of plates passing basic viability criteria, providing a systematic approach applicable to multi-well plate-based behavioral assays. Developmental toxicity (DT) endpoints showed high inter-laboratory concordance (84-100%), while DNT activity calls were more variable (68-84%); however, potency estimates for active compounds showed similar reproducibility (mean 3.2-fold for DNT, 3.5-fold for DT). Zebrafish LDTT detected 93% of DNT-positive compounds and 100% of DNT-negative compounds as a single whole-organism assay, demonstrating complementary performance to the 13-assay DNT in vitro battery (DNT-IVB; 86% sensitivity, 60% specificity). Integration with DNT IATA case studies demonstrated that multi-laboratory zebrafish data strengthen weight-of-evidence evaluations, with independent validation establishing high confidence in the flame retardants TMPP and BPDP. Together, these findings support positioning zebrafish larval behavioral assays as an efficient first-tier whole-organism NAM that bridges mechanistic in vitro screening and mammalian guideline studies, providing organism-level points of departure and prioritizing compounds for further DNT-IVB and in vivo testing within tiered regulatory DNT assessment frameworks.
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