Figure 1.

Study design. Female A/J mice were exposed to filtered air (sham), 3 concentrations of Tobacco Heating System (THS) 2.2 aerosol (6.7, 13.4, and 26.8 µg/l nicotine), or 3R4F cigarette smoke (13.4 µg/l nicotine). Additionally, male mice were exposed to filtered air or THS 2.2 aerosol (26.8 µg/l nicotine). The experimental period was preceded by 25 days of acclimatization. Interim dissections of subgroups of female mice were performed after 1, 5, and 10 months of exposure. Terminal dissections of the male and female mice were performed in months 15 and 18, respectively. At selected time points, the animals were allocated for analysis of the following endpoints: OECD toxicology endpoint analyses (mortality, hematological analysis, clinical chemistry analysis, and urinalysis), bronchoalveolar lavage fluid (BALF) analysis by flow cytometry and multi-analyte (cytokine/chemokine, growth factor) profiling, histopathological evaluation of respiratory and nonrespiratory tract organs, lung function tests, lung morphometry, lung tumor analysis, and systems toxicology analysis (transcriptomics, proteomics, and DNA sequencing). THS, tobacco heating system; CS, cigarette smoke; OECD, Organisation for Economic Co-operation and Development; BALF, bronchoalveolar lavage fluid.