IPPD biotransformation and oxidative stress associated with its metabolite mixtures

N-Isopropyl-N'-phenyl-1,4-phenylenediamine (IPPD) is the second most widely used p-phenylenediamines (PPDs) antioxidant in rubber products, following N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD). Although IPPD can be oxidized to IPPD‑quinone (IPPD‑Q) in the environment, it remains unclear whether this transformation occurs in mammals, and the potential adverse effects of IPPD metabolites are still poorly understood. In this study, using rat in vitro metabolic systems combined with suspect screening and non-target analysis, IPPD was rapidly metabolized, with 64.8% depletion in the Phase I system (t_1/2 = 46.0 min; CL_int = 43.7 μL min^-1 mg^-1 protein) and 98.5% depletion in the Phase II system, yielding 11 Phase I metabolites and eight Phase II conjugates. Ten of these metabolites were also detected in human in vitro metabolic systems. IPPD was enzymatically transformed into IPPD-Q, suggesting an endogenous quinone formation pathway. Among the identified metabolites, M12 and M17 showed stable temporal profiles and were further detected in urine from IPPD-exposed mice, suggesting their potential as candidate exposure biomarkers. Oxidative stress assays showed that IPPD incubation mixtures induced time-dependent responses in HepG2 cells, with MDA levels decreasing from 158.0 to 60.8 μM mg^-1 protein over 90 min, while superoxide dismutase (SOD) activity peaked at 9.5 U mg^-1 protein at 15 min and then declined. Exposure experiments further showed that IPPD-Q exhibited higher oxidative stress potential than IPPD. These findings clarify IPPD biotransformation, reveal an endogenous pathway for IPPD-Q formation, and provide new evidence for human exposure assessment and toxicological evaluation of PPDs.