Abstract The outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) has led to >2800 hospitalized patients and to >60 deaths in the US. The number of cases peaked between June and September 2019 with a subsequent reduction in trends since then. However, cases continue to occur, emphasizing the need to understand better the underlying mechanism(s) of EVALI. Patient data has led to the hypothesis that vitamin E acetate (VEA) in e-cigarettes can generate a toxic mixture that leads to EVALI. Recently, e-vapor generated from liquid containing VEA has been found to induce lung injury in mice. However, knowledge gaps remain and a need exists to investigate further this hypothesis. This proposal has assembled a team of experienced investigators that are capable of chemical, molecular, and toxicological assessments to provide mechanistic insights into EVALI. We have obtained the following preliminary data: A. VEA e-vapor generated from current sub-ohm vaping devises can produce a novel reactive oxygen species: ethyl peroxyl radical. B. In primary mouse alveolar macrophages, e-vapor extract is cytotoxic and leads to membrane blebbing. C. In RAW 264.7 cells, e- vapor extract increases cell membrane phosphatidylserine externalization. D. In RAW 264.7 cells, e-vapor extract activates caspase 3/7 mediated cell death that can be inhibited by the antioxidant n-acetyl cysteine. E. In mice, single cell RNASeq implicates a number of critical events including decreased macrophage transcripts that are regulated by interferon regulatory protein 8. The hypothesis-driven aims are: 1. Determine the chemical culprits that produce the pathological responses of EVALI. 2. Determine the mechanism of E-vapor induced cell death in alveolar macrophages, and 3. Determine the role of E-vapor-induced decreased interferon regulatory factor 8 in macrophage function. In the latter aim, the mechanisms by which corticosteroid therapy used in EVALI recovery will be investigated. Each aim considers alternative outcomes and strategies. In future studies, our approaches could be applied to flavored e-liquid toxicology. EVALI may have mechanisms that are common to other chemicals (e.g., acrolein or phosgene) known to induce acute lung injury in humans. Thus, the knowledge gained about EVALI pathology and therapy could be applied broadly to chemically-induced acute lung injury. Alternatively, EVALI may be a unique form of acute lung injury and a clearer understanding of its pathology will provide a mechanistic basis for the targeted strategies to treat this syndrome alone.