The zebrafish visual and acoustic motor response (VAMR) assay has the potential to add value to the developmental neurotoxicity in Vitro battery (DNT IVB)

Less than 150 chemicals have been evaluated for their potential to cause developmental neurotoxicity (DNT) using OECD rodent-based test guidelines. The DNT in vitro battery (DNT IVB), comprised of 17 human or rodent cell-based assays, is a proposed alternative. Recent work identified three false negative compounds that are developmentally neurotoxic in rodent or human studies but negative in the DNT IVB (5,5-diphenylhydantoin, maneb, nicotine). We hypothesized that multi-behavioral phenotyping in early-life stage zebrafish can complement the DNT IVB. Zebrafish embryos or larvae were acutely (40 min) or developmentally (1-5 days post-fertilization) exposed to 12 DNT positive chemicals 5,5-diphenylhydantoin, BDE-99, chlorpyrifos, haloperidol, hexachlorophene, ketamine hydrochloride, maneb, nicotine, PFOA, tebuconazole, trichlorfon, or triethlytin bromide or to 0.4% DMSO. Behavior was evaluated using the Visual and Acoustic Motor Response (VAMR) assay, comprised of 26 behavioral endpoints including visual and acoustic startle responses, non-associative habituation learning, and memory retention. Five proposed DNT negative compounds were also evaluated under both exposure paradigms. To fit concentration response data, generalized additive and linear mixed effects modeling strategies were combined with Fréchet distance calculations. Hit calls were identified such that inclusion of the zebrafish VAMR assay within the DNT IVB would potentially increase battery sensitivity without compromising battery specificity. Hits in the VAMR assay were typically detected at 1-4 orders of magnitude lower concentrations relative to the DNT IVB. Taken together, the zebrafish behavior-based VAMR assay can add value to the DNT IVB by increasing test battery sensitivity and enhancing potency detection.