Zebrafish behavioral assessment as a first-tier whole-organism NAM for developmental neurotoxicity: A...

Area of Science:

• Toxicology
• Neuroscience
• Developmental Biology
• New Approach Methodologies (NAMs)

Background:

• Developmental neurotoxicity (DNT) assessment requires validated, reproducible New Approach Methodologies (NAMs).
• Tiered testing strategies necessitate clearly defined roles for novel assays.
• Multi-laboratory validation is crucial for regulatory acceptance of NAMs.

Purpose of the Study:

• To evaluate the inter-laboratory reproducibility of the zebrafish Light-Dark Transition Test (LDTT) for DNT assessment.
• To assess the performance of LDTT as a whole-organism assay within a tiered testing framework.
• To compare LDTT performance with existing DNT in vitro batteries (DNT-IVB).

Main Methods:

• Multi-laboratory evaluation of the zebrafish LDTT across four independent facilities.
• Testing of 33 compounds, including known DNT toxicants and negatives, using standardized protocols.
• Implementation of a three-tiered quality control framework for data filtering.

Main Results:

• High inter-laboratory concordance for developmental toxicity (DT) endpoints (84-100%) and reproducible potency estimates for DNT (mean 3.2-fold).
• Zebrafish LDTT demonstrated high sensitivity (93% for DNT positives, 100% for negatives).
• LDTT data strengthened weight-of-evidence evaluations in case studies, validating specific flame retardants.

Conclusions:

• The zebrafish LDTT is a reproducible, efficient, whole-organism NAM suitable for first-tier DNT screening.
• LDTT complements in vitro assays and provides organism-level data for regulatory DNT assessment.
• Findings support the integration of zebrafish behavioral assays into tiered regulatory frameworks.