IN VITRO EVALUATION OF AIRWAY TOXICITY USING THE EPIAIRWAY ORGANOTYPIC IN VITRO HUMAN AIRWAY MODEL.

Recent REACH legislation has heightened the need for validated in vitro airway toxicity models. However, in vitro determination of airway toxicity potential is problematic since submerged monolayer cell cultures are not amenable to dosing of vapors or water insoluble chemicals. The EpiAirway model is an organotypic air-liquid interface culture produced from normal human airway epithelial cells. EpiAirway more adequately reproduces in vivo airway epithelial morphology and is more amenable to dosing since the apical surface is exposed to the atmosphere. Here we describe an approach to validate the EpiAirway model for airway toxicology applications. Two dosing protocols were developed and evaluated: 1) Direct application of test chemicals in aqueous or corn oil vehicles, and 2) a vapor cup method for dosing volatile chemicals. Dose response experiments were conducted to determine EC50 concentrations for loss of culture viability, transepithelial electrical resistance or release of inflammatory mediators. EC50 concentrations were correlated with OSHA established Immediately Dangerous to Life and Health (IDLH) concentrations. Direct application was found to perform well for chemicals with IDLH values up to ~1,000 mg/m3. A prediction model based on EC50 (culture viability) after direct application was established for 15 chemicals. A correlation coefficient of r2=0.79 was obtained for the entire chemical set. Alkyl amines were found to be over-predicted by the direct application protocol. With alkyl amines removed from the data set the correlation improved to r2=0.98. Direct application of chemicals with IDLH values above ~1,000 mg/m3 was generally not possible. A vapor cup dosing protocol was developed for these chemicals. To-date vapor cup results for 11 volatile chemicals including a diverse range of chemical classes produced a correlation coefficient of r2 = 0.83 with respect to in vivo IDLH values. Thus, the prediction models appear to provide promise for in vitro determination of airway toxicity. Future work will validate these models with larger chemical sets.

AIR-100, Airway irritation, EC75, EpiAirway™, IL-8, Immediately Dangerous to Life or Health (IDLH), In Vivo-InVitro Correlations, Permissible Exposure Limits (PEL), Vapor cup dosing, Volatile chemicals

Acetic acid, Acetone, Cyclohexene, Diisopropylamine, Ethyl Acetate, Ethyl formate, Formaldehyde, Isopropanol, P-Xylene, Tetrachloroethylene, Toluene