The aim is to demonstrate the feasibility of TT21C in vitro methods as part of an overall assessment framework for next generation products (NGP).

TT21C methodologies can be used to assess harm reduction potential of NGPs using human-derived cellular systems and biological responses. The potential harm reduction potential of three different NGP products were compared to the reference cigarette (3R4F) in a series of in vitro assays. Either whole smoke/aerosol or phosphate buffered saline (PBS) trapped smoke/aerosol were used. The products investigated were the Kentucky reference cigarette (3R4F, 1.8 puffs/ml), a tobacco heated product (THP), a hybrid product (HYB) and a myblu™ e-cigarette (Tobacco Flavour 1.6 % Nicotine). The 3R4F and THP were smoked using the HCI Intense smoking regime. HYB and myblu™ were vaped according to CORESTA Recommended Method No. 81 (CRM 81). Four puffs/ml of PBS was used for all NGPs.

Smoke/aerosols were captured in PBS to enable the use of in vitro systems where direct exposure to smoke/aerosol was not possible. Chemical characterisation was conducted on whole smoke/aerosol (WHO TobReg 9) and on aerosol/smoke PBS solutions to measure nicotine and 8 carbonyls.

Regulatory accepted in vitro assays (NRU, Ames, IVMNT) were employed for testing whole smoke/aerosol from the different platforms. All assays indicated reduced cytotoxic and genotoxic activity of the THP compared to 3R4F. There were limited to no effects observed in each assay for HYB and myblu™. Test samples were also analysed in several TT21C assays to provide a wider mechanistic understanding, including cardiovascular disease (scratch wound), COPD, tumour promotion (Bhas cellular transformation assay) and cellular health (high content screening); 3D reconstituted bronchial epithelia with repeated exposure; multicellular/organ profiling (DiscoverX) and developmental toxicity. The results of all assays indicated limited to no toxicity for myblu™ aerosol or extracts. Based on the tests conducted the overall ranking in terms of the most active product was 3R4F > THP > HYB ≥ myblu™.