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The use of electronic nicotine delivery systems, such as e-cigarettes and heated tobacco products (HTPs), is increasing, but knowledge of their short and long-term toxicological effects remains limited. Here, aerosols generated from an e-cigarette using a flavour-free e-liquid base, both with and without nicotine, an HTP, and a conventional cigarette were characterised for the production of polycyclic aromatic hydrocarbons (PAHs), carbonyls and volatile organic compounds (VOCs). Furthermore, extracts from vapour and smoke were generated, and their acute toxicity was assessed in human lung epithelial cells and fibroblasts. Cigarette smoke contained significantly more toxic compounds and induced the highest degree of toxicity in all the tested cell lines, followed by the HTP, and then the nicotine containing e-cigarette. Notably, the nicotine containing e-cigarette produced similar levels of formaldehyde as the HTP and cigarette smoke, and caused a greater decrease in cell viability in primary lung fibroblasts compared to the nicotine-free e-cigarette. Although the HTP aerosol contained lower levels of toxicants than cigarette smoke, some VOCs specific to HTPs were detected. More independent research is needed to identify toxicant-specific production in emerging nicotine delivery systems and their potential health impacts to better inform policy makers, health care providers and the general public.