A Proof-of-Concept for Safety Evaluation of Inhalation Exposure to Known Respiratory Irritants Using In Vitro and In Silico Methods

There is increased interest in developing non-animal test systems for inhalation exposure safety assessments. However, defined methodologies are absent for predicting local respiratory effects from inhalation exposure to irritants. The current study introduces a concept for applying in vitro and in silico methods for inhalation exposure safety assessment. Three in vitro systems, representing the upper (MucilAir™ - nasal epithelial tissue) and lower (A549 cells and human precision-cut lung slices) human respiratory regions, were exposed to six respiratory irritants. These irritant exposures were conducted as liquid droplets, aerosol, or vapors, and samples were collected over 24 h. Cytotoxicity, cytokine release, epithelial resistance, oxidative stress, and mitochondrial membrane potential were measured. To determine the human relevance of in vitro exposures, airway surface depositions were predicted by simulating airborne concentrations equivalent to the Cramer class III inhalation threshold of toxicological concern limit of 0.47 mg/person/day using an in silico model. A > 100-fold margin of exposure was calculated comparing lowest concentrations showing in vitro effects to in silico simulated values. While further studies are needed, this manuscript presents a basic requirement for employing non-animal methods to inform inhalation exposure safety assessments by combining in vitro and in silico assays.