Assessment of cytotoxicity and mechanistic insights from a combination of three independent anatomical regions within the respiratory tract using in vitro 3D human models following exposure to a 1R6F reference cigarette

World Congress on Alternatives and Animal Use in the Life Sciences (WC13) 2025 -- New approach methodologies (NAMs) are being implemented for inhalation toxicity to deliver improved safety assessment and hazard identification. There is an increased focus to investigate products in more physiologically relevant environment (e.g., continuous flow versus static). 3D models are a relevant method for analysis of toxicity and modelling downstream effects such as inflammation and additionally complying with the principles of 3Rs. This study utilized models from Epithelix Sarl (Switzerland) and included MucilAir™, SmallAir™ and AlveolAir™ in their response to whole smoke from a 1R6F Kentucky reference cigarette. In this study, we used the lactate dehydrogenase (LDH) release, WST-8 to assess toxicity. Trans-epithelial electrical resistance (TEER), cilia beat frequency, a pro-inflammatory cytokine panel and RNA analysis were performed to provide molecular insights. Exposure of MucilAir, SmallAir and AlveolAir to whole smoke resulted in different levels of viability and different mechanistic insights. This study was a proof of concept suggesting that different anatomical locations of 3D human lung models can be used for safety assessment and for elucidating pathways of cytotoxicity through mechanistic insights.