In Vitro approaches for assessing respiratory toxicity

Risk assessment and management relies on approaches that can accurately and efficiently predict the toxicity of chemicals in humans. Inhalation is a major route by which exposure to substances can occur, and is an area where resources have been dedicated to optimizing relevant in vitro approaches based on human biology. In vitro approaches that employ proper controls, are reproducible, and simulate physiological conditions have been developed and can be used to test substances and concentrations that are relevant to human exposures. Presented here is an example of an in vitro testing strategy using BEAS-2B cells (a human bronchial epithelial cell line) as a model to assess the ability of silanes to cause portal-of-entry effects on the human respiratory tract. The cells were exposed to various concentrations (0.72ppm, 25ppm, and 85ppm) of triethoxysilane vapor—a GHS category 2 inhalation toxicant based on rat LC50 (lethal concentration, 50%) data—at the air-liquid interface using a capillary dosage unit coupled to a VITROCELL 6/4 exposure module. A significant concentration-dependent decrease in cell viability (resazurin-based assay) and increase in cytotoxicity (lactate dehydrogenase assay) was observed after exposure to the triethoxysilane and nitrogen dioxide (positive control) as compared to clean air (negative control). A significant increase in expression of inflammatory markers [interleukin (IL) 4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumor necrosis factor-alpha (TNF-α)], determined by Meso Scale Discovery technology, was observed at 25ppm. Work is underway to test additional controls (Triton X-100 and sodium chloride) and silanes (with varying carbon-chain length) to assess the advantages of using a 2D cell line (BEAS-2B cell) versus a 3D human reconstructed tissue model (MucilAir™, Epithelix). This project has included regular discussions and information-sharing with experts from industry and regulatory agencies regarding choice of in vitro system, test materials, biological end points, and appropriate controls relevant to assays and test materials. These multi-sector discussions have helped facilitate the uptake of robust in vitro methods and reduce the use of animals in testing.