Toxicity assessment of 6PPDQ and Its hydroxy metabolites in fish cell lines and coho salmon

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPDQ), a transformation product of the tire antidegradant 6PPD, is associated with acute mortality in sensitive salmonids. Although hydroxylation is typically considered an initial detoxification step prior to conjugation, the toxicity of specific hydroxylated metabolites remains poorly understood. In this study, we evaluated the cytotoxicity of 6PPDQ, quinone ring-hydroxylated 6PPDQ-1-OH, and phenyl ring-hydroxylated p‑hydroxy-6PPDQ using three salmonid-derived cell lines (coho salmon CSE 119, rainbow trout RTG 2, and Chinook salmon CHSE 214) and complemented these data with in vivo acute toxicity tests in juvenile coho salmon. In vitro, 6PPDQ showed the highest toxicity in coho salmon cells, moderate toxicity in rainbow trout cells, and no detectable effect in Chinook salmon cells. 6PPDQ-1-OH exhibited markedly reduced cytotoxicity across all cell lines. In contrast, p‑hydroxy-6PPDQ was more potent than the parent quinone in all cell lines, with pronounced interspecies differences in sensitivity. In vivo, 6PPDQ-1-OH showed no mortality at a nominal concentration of 1000 ng/L, whereas p‑hydroxy-6PPDQ induced acute lethality in coho salmon, with a 24 h LC₅₀ of 113 ng/L. These results demonstrate that hydroxylation is not uniformly detoxifying and that the position of hydroxylation critically influences toxicity. Our findings highlight the importance of metabolite-specific evaluation for understanding the toxic effects of tire road wear particle-derived chemicals.