Toxicological Research to Assess Bioaccessibility and Biological Effects of Recycled Tire Crumb Rubber Using In Vitro and In Vivo Testing Approaches

摘要

The National Toxicology Program conducted in vitro and 14-day in vivo studies of tire crumb rubber (TCR) to enhance understanding of potential health impacts of exposure to chemicals released from TCR, and to evaluate (1) utility of different experimental models for characterizing toxicity, (2) routes of exposure that may result in systemic exposure, and (3) bioaccessibility of TCR constituents. Multiple lots of fresh recycled TCR were received from California EPA and combined into one test lot for use in these studies. The lot was characterized using microscopy, elemental composition, metal analysis, as well as liquid (LC-MS) or gas chromatography coupled with mass spectrometry. In vitro studies were conducted with human-derived skin, lung, small intestinal and liver cell lines using TCR-conditioned media (TCR-CM). TCR was incubated in cell type-specific culture media for different durations and temperatures followed by sterile filtering to generate TCR-CM for cell exposures. Cytotoxicity was observed with all cell lines, except liver, which was dependent upon concentration as well as the duration and temperature of media conditioning with TCR. Conversely, TCR incubated in PBS or artificial lung fluid was not cytotoxic. Untargeted LC-MS was used to characterize the chemical composition of TCR-CM. In vivo 14-day studies in female B6C3F1/N mice were conducted by administering size-fractionated TCR by oral gavage, dosed-feed or by housing on TCR mixed-bedding. Traditional measures of toxicology testing were evaluated and showed no toxicologically relevant changes by the routes tested. Using a LC-MS metabolomics analysis approach, urine and plasma were evaluated for evidence of exposure. Principle component analysis of untargeted data was unable to differentiate treated and control groups, likely due to low exposure levels and high interanimal variability. Compounds potentially originating from TCR were tentatively identified in urine and plasma from gavage animals.