In vitro studies have been widely used to support toxicological evaluations of chemicals and complex mixtures including cigarette smoke. In this study a variety of test matrices from tobacco and nicotine delivery products were assessed against a 3R4F reference cigarette using the in vitro micronucleus assay (IVMN). Assays were conducted in general accordance to OECD Guideline 487 and ICH S2 (R1) guidance. Samples were also assessed using the mouse lymphoma assay (Part 1).

3R4F total particulate matter (TPM) was first assessed with CHO, V79 (both rodent) and TK6 lymphoblastoid (human) cell lines with 3 h exposures ±S9 metabolic activation and extended -S9 treatments with/without a 1.5-2 cell cycle length recovery period at doses up to 500 µg/mL. CHO, V79 and TK6 cells gave varied positive responses, with V79 being most responsive. The extended recovery treatment increased assay sensitivity for CHO and V79 cells; this was less clear in human TK6 cells. V79 cells were taken forward for further assessments.

3R4F TPM was compared against pad-collected aerosol matter generated from a commercial electronic nicotine delivery system (ENDS), a commercial e-liquid, and TPM from a commercial tobacco-heating product (THP) using the same treatment schedules described above.

Across all treatment regimens with V79 cells, clear negative responses were observed for the e-liquid, ENDS and THP samples, while 3R4F elicited a clear positive response. The most potent 3R4F responses were observed following extended treatment -S9 with recovery, suggesting this to be a more appropriate treatment schedule for the assessment of tobacco and nicotine product test matrices. Based on the results of this study the IVMN assay can be used effectively for the assessment of these test matrices.