| Sumario: | Public health concern for playing on synthetic turf fields with crumb rubber infill has increased in recent years. Crumb rubber manufactured from recycled automobile tires contains potentially toxic and carcinogenic substances, and, with over 12,000 synthetic turf fields in the United States, the potential for exposure is widespread. The National Toxicology Program (NTP) conducted research to improve the understanding of potential human exposure to crumb rubber and its biological activity. The objective of the in vitro studies as described in this report was to determine the cytotoxicity of extracted crumb rubber using cultured human skin-derived keratinocytes (HaCaT cells) and peripheral lung (HPL-1D) cells to reflect potential cellular targets of toxicity from dermal and inhalation routes of exposure, respectively. Crumb rubber (100 mg/mL) was incubated in cell type-specific culture medium for 3 hours or 1, 4, or 7 days at ambient, 37°C, or 60°C temperatures to allow for the extraction of chemicals from the crumb rubber into the culture medium. Crumb rubber-conditioned medium (CRCM; 1X, generated from 100 mg/mL crumb rubber) was then sterile-filtered and serially diluted to 0.5, 0.25, 0.125, and 0.0625X concentrations for cell exposures. Control medium was simultaneously incubated without crumb rubber at matched times and temperatures. A CRCM-induced decrease in cell viability (i.e., cytotoxicity) was observed for both HaCaT and HPL-1D cell lines after 24-hour exposures. For both cell types, cytotoxicity was most pronounced after incubation of crumb rubber at 60°C, but was also observed at 37°C and ambient temperatures. In addition, CRCM was cytotoxic to human small intestinal (FHs-74-Int) cells, which were used to reflect a potential cellular target from ingestion, but not human hepatocytes (HepaRG cells). To determine if the observed cytotoxicity of CRCM was possibly due to the presence of serum proteins in the culture medium during extraction, crumb rubber was incubated in phosphate-buffered saline (PBS) or artificial lung fluid (ALF), sterile-filtered, diluted in culture medium, and then evaluated for cytotoxic effects. ALF, which is similar to pulmonary surfactant, is more physiologically relevant to the lung than the culture medium used for HPL-1D cells. Crumb rubber-conditioned PBS and ALF were much less cytotoxic to the HPL-1D lung cells than CRCM. This suggests that the cytotoxic effect of CRCM in vitro might not be biologically relevant in vivo or to crumb rubber exposures in humans. Samples of CRCM and crumb rubber-conditioned PBS or ALF were analyzed by non-targeted ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). Principal component analysis of the data showed segregation of chemical features present in the CRCM from the crumb rubber-conditioned PBS and ALF. Chemical compounds associated with the vulcanization of rubber were extracted from the crumb rubber. Among the chemicals identified using non-targeted analysis were 2-mercaptobenzothiazole, N,N'-diphenylguanidine, and 1,2-benzisothiazoline-3-one. By providing in vitro data regarding some of the extractable chemical constituents of crumb rubber, in conjunction with chemical characterization and in vivo testing, these studies will contribute to our understanding of potential exposure and biological activity of crumb rubber in humans.
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