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Zebrafish Behavioral Assessment as a First-Tier Whole-Organism NAM for Developmental Neurotoxicity: A Multi-Laboratory Evaluation

Lisa Truonga*, Jui-Hua Hsiehb*, Lee Ellisc, Michael Morashc, Oihane Jakad, Arantza Murianad Beatriz Molina Martínezd, Ana del Pozod, Valentina Schiavonee, Vincenzo Di Donatoe, Claudia Miguel Sanze, Robyn L Tanguaya, Ellen Hesself, Kristen Ryanb

a Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, US
b Division of Translational Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, US
c National Research Council of Canada, Halifax, NS, Canada
d BBD BioPhenix SLU - Biobide, San Sebastian, Gipuzkoa, Spain
e ZeClinics SL, Barcelona, Spain
f Centre for Health Protection, National Institute for Public Health and the Environment, Utrecht, Netherlands
*These authors contributed equally to this work.

DOI: https://doi.org/10.22427/DATA-500-107-004-000-7

Highlights

• Novel QC framework removes plates with irregular behavior of untreated larvae
• Multi-plate zebrafish behavioral assay shows reproducible results across four labs
• Multi-lab zebrafish behavioral assay shows high sensitivity and specificity for DNT
• Whole-organism zebrafish behavioral data strengthen OECD DNT case studies
• Zebrafish behavioral assay supports use as a first-tier NAM for DNT assessment

Publication

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.

Data

Supplemental Tables

Supplemental Data & Figures

The supplemental data provide plate-level, endpoint-level, and potency-level datasets as well as movement-over-time plots, supporting all analyses presented in the study. Only data from plates that passed all quality-control (QC) steps are included.

Movement-Over-Time Plots

This download contains per-plate movement-over-time visualizations.

Each panel displays the median distance moved per minute for viable larvae treated with vehicle control or with each test concentration. Files are organized by laboratory (e.g., Lab-A_movement_plate.pdf) as well as are organized by chemical for all laboratories (e.g., Lab-ABCD_movement_plate.pdf). The chemical code to chemical name table is available in the Supplemental Data & Figures.

Plate Data (Raw Data)

This download contains the raw plate-level zebrafish LDTT data that passed all QC criteria. Includes distance moved per minute data and developmental toxicity observations for each embryo. Files are stored in RDS (or TSV) format and organized by laboratory. One XLSX file includes all four-laboratory data.

Endpoint Data

This download contains calculated analytical endpoints (DNT and DT) for each plate. Files are provided in RDS format (or TSV) format and organized by laboratory. Two XLSX file includes all four-laboratory DNT and DT data.

Potency Data

This download contains benchmark concentration (BMC) and activity concentration at cutoff (ACC) results for both the zebrafish LDTT and the DNT-IVB assays. Data include full potency outputs and intermediate summary datasets.