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 have
demonstrated that 6PPD-quinone toxicity occurs at much lower concentrations
than the 6PPD parent molecule, which is consistent with available in vivo 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 p-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 those of 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.