Benchmark Dose Modeling Approaches for Volatile Organic Chemicals using a Novel Air-Liquid Interface In Vitro Exposure System

Inhalation is the most relevant route of volatile organic chemical (VOC) exposure; however, due to unique challenges posed by their chemical properties and poor solubility in aqueous solutions, in vitro chemical safety testing is predominantly performed using direct application dosing/submerged exposures. To address the difficulties in screening toxic effects of VOCs, our cell culture exposure system permits cells to be exposed to multiple concentrations at air-liquid interface (ALI) in a 24-well format. ALI exposure methods permit direct chemical-to-cell interaction with the test article at physiological conditions. In the present study, BEAS-2B and primary normal human bronchial epithelial cells (pHBEC) are used to assess gene expression, cytotoxicity, and cell viability responses to a variety of volatile chemicals including acrolein, formaldehyde, 1,3-butadiene, acetaldehyde, 1-bromopropane, carbon tetrachloride, dichloromethane, and trichloroethylene. BEAS-2B cells were exposed to all the test agents, while pHBECs were only exposed to the latter four listed above. The VOC concentrations tested elicited only slight cell viability changes in both cell types. Gene expression changes were analyzed using benchmark dose (BMD) modeling. The BMD for the most sensitive gene set was within one order of magnitude of the threshold-limit value reported by the American Conference of Governmental Industrial Hygienists, and the most sensitive gene sets impacted by exposure correlate to known adverse health effects recorded in epidemiologic and in vivo exposure studies. Overall, our study outlines a novel in vitro approach for evaluating molecular-based points-of-departure in human airway epithelial cell exposure to volatile chemicals. Overall design: We developed a cell culture exposure system (CCES) that permits cells to be exposed to multiple concentrations of test agent at air-liquid interface (ALI) in a 24-well format. ALI exposure methods permit direct chemical-to-cell interaction at physiological conditions, providing a more environmentally realistic airway exposure condition, as compared to submerged cultures. We tested eight widespread volatile organic chemicals (VOCs) in BEAS-2B cells with four of those test agents repeated in primary human bronchial epithelial cells (pHBECs). To stay within relevant concentrations and below overt cytotoxicity, the highest concentration tested was targeted as 10-fold higher than the threshold for human occupational exposure limits as determined by the American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value (TLV) documentation. The primary endpoint focused on using TempO-Seq to identify changes in gene expression at both the gene and aggregate gene set level to determine the biological pathway altering concentration (BPAC) of a particular test agent.