Alternative model for repeated dose inhalation toxicity using precision-cut lung slices

Testing inhalable substances regarding respiratory adverse effects in accordance with the three R principles requires appropriate alternatives for animal inhalation studies. Precision-cut lung slices (PCLS) are an alternative widely used for testing of adverse outcomes for the respiratory tract. Yet, PCLS have been used mostly for acute respiratory toxicity questions. To assess PCLS suitability for long-term toxicity studies, e.g., testing slowly metabolized inhalable chemicals, PCLS were long-term cultivated (>=14 d). For repetitive exposure rat PCLS were treated on three consecutive days with different concentrations of a model compound (TritonX-100) and compared to single and double exposure. LDH and WST-1 assay were used to assess vitality. Additionally, structural and functional variations were analysed by histopathology and bronchoconstriction over the 14-days period. Vitality remained constant over time with slight decreases towards day 14. Histopathology showed no fibroblasts proliferation, but preservation of alveolar structure, bronchiolar epithelium and smooth musculature. Bronchoconstriction was measureable for 14 days, with slightly decreased sensitivity (EC50_1d=8.22x10-8M vs. EC50_15d=4.70x10-7 M methacholine). Repeated exposure did not influence the sensitivity of the lung tissue (EC50_1d=76 M to EC50_3d=80 with constant vitality.