Virtual effect-directed analysis of granulated rubber identifies bioactive chemicals and distinct hazard profiles

This study applied a virtual effect-directed analysis (vEDA) approach, integrating effect-based analysis and chemical screening, to identify bioactive compounds in rubber infill from artificial turf. Bioreporter assays targeting diverse toxicological endpoints were selected to detect a wide range of potential endocrine-disrupting and genotoxic compounds. Of 21 samples, all except one showed aryl-hydrocarbon receptor (AhR) activity (14-31,400 ng benzo[a]pyrene equivalents/g), four induced p53 activity (0.04-0.86 µg actinomycin D equivalents/g) and two showed estrogen receptor α (ERα) activity (530 and 1020 pg estradiol equivalents/g). Chemical analysis quantified up to 87 polycyclic aromatic compounds (PAC) and gas chromatography high-resolution mass spectrometry-based suspect screening yielded 281 tentative identifications. Annotation with bioassay activity data from databases and predictive models revealed 29 AhR-, 32 ERα- and 18 p53-active compounds. Univariate analysis was used to prioritize compounds for further chemical and toxicological confirmation. Eighteen AhR agonists were confirmed, contributing 0-98% to the observed AhR activity in the samples. Phenylamine additives, detected at high concentrations, exhibited low AhR activating potency and contributed < 1%. In contrast, methylated chrysene isomers elicited relatively high potencies and contributed substantially (≤65%) to the observed AhR activity. N-Isopropyl-N'-phenyl-p-phenylenediamine (IPPD) was confirmed as p53 active and explained ∼50% of the observed activity in the most p53-active sample. Styrene-butadiene rubber (SBR) showed higher AhR- and p53 activities and concentrations of quantified compounds than the alternative materials. The study highlights differences in chemical hazards among rubber infill materials and demonstrates the utility of vEDA as an early-warning tool for identifying compounds of concern.