Fig. 3: Workflow for screening and data analysis of the NeuriTox test.

During the screening, each NTP80 compound was tested at 10 concentrations (plus solvent control), starting at a maximum concentration of 20 μM and then in three-fold dilutions. Altogether, the screen was performed three times (independent biological replicates). Curve fitting was performed for neurite area (NA) and viability (V). If NA or V was neither significantly different from solvent control nor altered to more than 20%, the compound was classified as inactive. Otherwise the compound was considered to be active. If active compounds affected NA at more than one concentration that did not affect V, they were classified as “hits”. These were retested independently in an adjusted concentration range with subsequent curve fitting and EC₅₀ calculation. If the EC₅₀ ratio of V/NA was ≥ 4, the compound was classified as a “specific (developmental) neurotoxicant”. Otherwise it was classified as “cytotoxic”.