Utilizing Multiple Behavioral Endpoints to Identify Negative Control Chemicals in a Larval Zebrafish Behavior Assay

Identifying reliable negative control compounds is essential for determining the sensitivity and specificity of screening assays. However, well-characterized negative controls for developmental neurotoxicity behavioral assays in larval zebrafish (Danio rerio) are lacking. This study evaluated nine chemicals with no reported evidence of mammalian developmental neurotoxicity, and a positive control (fluoxetine) for developmental and neurodevelopmental (i.e., behavioral) toxicity in zebrafish. Embryos were exposed to each chemical (≤100 µM) during development, 0-5 days post-fertilization (dpf), then assessed as larvae (6 dpf) using a locomotor behavior light-dark transition test. Behavior was analyzed using two methods: (1) the traditional method, comparing the average total distance moved, and (2) a 13-endpoint approach analyzing 13 aspects of the locomotor profile. Results showed that ibuprofen, omeprazole, and fluoxetine induced developmental toxicity (teratogenesis), with fluoxetine also causing behavioral neurotoxicity. Behavioral effects of developmental exposure to selegiline hydrochloride depended on the analysis method. Exposure to the other six chemicals (D-mannitol, glycerol, L-ascorbic acid, metformin hydrochloride, saccharin, and sodium benzoate), as well as ibuprofen or omeprazole, did not produce behavioral effects using either analysis method. Identifying negative control chemicals is essential for evaluating behavioral alterations precipitated by unknown substances and will assist with screening new chemicals for neurodevelopmental toxicity.