P30-13 Respiratory Toxicity Risk-Based Decision Making Using Organotypic Lung Models

Inhaled chemical toxicity is assessed using lung organotypic models (OECD, 2022; US EPA, 2023), but the lack of standardized guidelines limits non-animal alternatives. We are developing standardized in vitro aerosol screening protocol to support an IATA for inhalation toxicity. We evaluated acute toxicity and point of departure for chemicals with known inhalation risks, using EC25 values in 2D and 3D human airway models and a 3D rat model across two labs with harmonized protocols. Toxicity was assessed for 10 chemicals spanning all UN GHS acute inhalation hazard categories in primary human bronchial cells via direct application and assessed 24-hour post-dose. Chemicals were tested in 2D monolayers and apically in 3D human and rat EpiAirway™ tissues. Experiments were conducted at Charles River (CRL) and MatTek (MTK) for inter-lab comparison. Membrane integrity (TEER 3D only), cell viability (alamarBlue all) and cytokine release were measured. EC25 values were interpolated, and statistical analysis (drc package R) compared slopes and EC25/EC50 parameters with significance set at p<0.05 and a >2.5-fold difference. Results were analyzed for inter-lab reproducibility. 2D model showed high reproducibility with EC25 values within a 2-fold range for 9/10 chemicals across labs. Monolayers were more sensitive than 3D-human tissues for 9/10 chemicals with sensitivity varying by chemical. 2D and 3D models reliably identified toxic and sub-toxic levels. EC25 values for 8/10 chemicals were within 2.5-fold across labs (3D-human model), demonstrating reproducibility. TEER EC25 values <3 mg/mL predicted toxic materials (Categories 1-3), while values >3 mg/mL predicted Category 4, and values exceeding the highest tested concentration indicated Category 5, alamarBlue results were similar but slightly less sensitive. 3D-Rat model showed reproducibility with 7 (alamarBlue) and 8 (TEER) EC25 values within 2.5-fold across labs. EC25 values were slightly higher in rat than human model. Rat TEER EC25 values <5 mg/mL indicated hazardous materials (Categories 1-3), while values >5 mg/mL identified non-hazardous materials (Categories 4-5). A panel of cytokines were analyzed and showed model and chemical differences in inflammatory response. The 2D model was used for dose selection for 3D testing and reproducibility between labs was achieved with harmonized protocols. The rat model allowed for translation between rodent and human outcomes and supports mathematical modelling for in vivo predictions. Human EpiAirway™ protocol shows promise for acute inhalation hazard classification and supports the development of an IATA. This study is part of a larger program incorporating repeat-dose studies, aerosol vs. liquid exposure, and donor variability to develop a predictive model for aerosol chemical inhalation risk.