Fig. 2

The three experimental phases. Phase 1 aimed at defining non-cytotoxic and very low toxic (IC₂₀/100, IC₅), and moderately toxic (IC₂₀) concentrations, compared to solvent control (0.1% DMSO) at the respective time point for each chemical. Goal of Phase 2 was to define for each individual chemical, for each time point (i.e., 3 days and 14 days) and for each analysed DNT endpoint (i.e., synaptogenesis, neurite outgrowth and BDNF levels), the Lowest Observable Adverse Effect Concentrations (i.e., LOAEC-syn, LOAEC-neu and LOAEC-bdnf), based on analysis of statistical significance. During this phase, treatments with single chemicals at the concentrations defined in Phase 1 (i.e., non-cytotoxic (IC₂₀/100), very low toxic (IC₅) and moderately toxic (IC₂₀)) were performed to assess their effects on synaptogenesis, neurite outgrowth and BDNF levels. During Phase 3, treatments with single chemicals and mixtures at the LOAECs concentrations defined at the end of Phase 2 were performed to assess possible combined effects, assessing the same DNT-specific endpoints as described in Phase 2