Phenotypic Profiling of 6PPD, 6PPD-Quinone, and Structurally Diverse Antiozonants in RTgill-W1 Cells Using the Cell Painting Assay

6PPD-quinone, a degradation product of the rubber antiozonant 6PPD that is frequently added to tires, has previously been identified as a causative agent of urban runoff mortality syndrome in salmonids. Previous high-throughput phenotypic profiling (HTPP) studies using the Cell Painting assay in the RTgill-W1 rainbow trout cell line has demonstrated that 6PPD-quinone toxicity occurs at much lower concentrations than the 6PPD parent molecule, which is consistent with available <i>in vivo</i> toxicity data in rainbow trout. Current research efforts include identifying alternative antiozonant compounds to potentially replace 6PPD in tire manufacturing. To fill bioactivity data gaps for potential 6PPD alternatives, 18 compounds including other substituted <i>p</i>-phenylenediamines (PPD) and PPD-quinones were assayed using HTPP in RTgill-W1 cells. 7PPD-quinone and 77PD-quinone produced changes in cellular phenotype similar to 6PPD-quinone at comparable concentrations. IPPD-quinone produced changes in cellular phenotype at higher concentrations than 6PPD-quinone, with a phenotypic profile that was most similar to its parent molecule IPPD. These findings suggest that 7PPD-quinone and 77PD-quinone may exhibit similar effects in rainbow trout, and potentially other 6PPD-quinone sensitive salmonids. In contrast, IPPD may be less toxic to salmonids than 6PPD, given the relative lack of bioactivity of IPPD-quinone compared to 6PPD-quinone.