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The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under "desert" or "tropical" conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.