Translating Zebrafish Science into Regulatory Utility: A Critical Gap Analysis of OECD Test Guidelines

The zebrafish is formally incorporated into several OECD Test Guidelines for chemical hazard and risk assessment, including TG 203, 210, 212, 215, 229, 230, 234, 236 (FET), and 250 (EASZY), with zebrafish-specific guidelines (ZEOGRT, iFEDT) under development. Related non-zebrafish guidelines addressing endocrine disruption, TG 240 (Japanese medaka) and TG 248/XETA (Xenopus), offer complementary but non-zebrafish coverage. Despite this expanding TG portfolio, significant research and methodological gaps continue to limit the regulatory utility, mechanistic depth, and predictive accuracy of zebrafish-based testing. This critical review systematically evaluates the principal gaps across existing zebrafish-relevant OECD TGs, including the chorion permeability barrier in TG 236, insensitivity to neurotoxicants and metabolically activated compounds, absence of standardized behavioral and molecular endpoints, incomplete thyroid axis coverage, unresolved mixture toxicity guidance, lack of validated omics interpretation frameworks, high inter-laboratory variability, and the non-mandatory status of developmental neurotoxicity testing. It further critiques the slow integration of AOP logic into TG structure, the absent regulatory framework for nanotoxicology and epigenetic endpoints, and methodological shortfalls in chronic and multigenerational testing. Collectively, these gaps constrain the capacity of OECD zebrafish TGs to meet the demands of contemporary chemical safety regulation under REACH, the EU Chemicals Strategy for Sustainability, and global harmonization efforts. Targeted recommendations are provided for each gap category to position zebrafish as a fully integrated, mechanistically credible regulatory test organism.