Integrated in vitro multi-endpoint framework for assessing 6PPD-Quinone cytotoxicity in fish and mammalian cell lines

The tire wear-derived quinone N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-Q) has attracted global attention due to its pronounced toxicity across diverse biological systems. To provide a quantitative evaluation of 6PPD-Q toxicity, we performed integrated cellular and phenotypic profiling of 6PPD-Q exposure across five cell lines: three fish-derived (PCK, CIK, RTG-2) and two mammalian cell lines (CHO-K1, Neuro-2a). Beyond conventional MTT viability assays, LDH release measurements, morphological analysis, apoptosis quantification, and cell cycle profiling, quantitative analysis of toxicity was achieved through two complementary scoring frameworks-phenotype-level Network Perturbation Amplitude (pNPA), calculated from multi-endpoint phenotypic perturbations, and the Relative Biological Impact Factor (RBIF)-enabling objective ranking of cytotoxicity and phenotypic outcomes. Under the tested in vitro conditions and with the cell lines used, fish-derived cells (PCK: yellow croaker kidney and RTG-2: rainbow trout gonad) generally displayed greater susceptibility than the mammalian cell lines (CHO-K1: hamster ovary and Neuro-2a: mouse neuroblastoma) as evidenced by pNPA values derived from toxicity data including reduced viability, elevated apoptosis rates, and distinct cell cycle arrest patterns, although tissue-origin differences remain a major limitation. This study advances the understanding of 6PPD-Q toxicity and reveals differential cellular effect mechanisms. Critically, it provides a quantitative in vitro framework for comparing 6PPD-Q-induced phenotypic perturbations and identifying dominant cytotoxic endpoints. Because the tested concentrations exceeded environmentally relevant levels, the findings should not be directly extrapolated to real-world ecological risk assessment.