Abstract: Modern oral tobacco nicotine products (ONP) are available in tobacco and other flavors. Traditional in vitro assays are not always able to differentiate toxicological impact of the different flavor profiles. The objective of this study was to assess the mechanisms and mode of action utilizing the stem cell- based reporter assay ToxTracker to determine if different flavors of oral products can be differentiated and if they have different reporter gene induction profiles. The American style loose moist reference product (CRP 2.1) and 4 different flavored (traditional, mint, white and wintergreen) commercially available ONP were extracted with DMSO and CAS. Concentrations up to 1.6 mg/mL of the extract were tested in each of the six reporter cell lines (+/- S9) of the ToxTracker assay. Following a 24hr incubation, reporter gene (GFP) induction and cytotoxicity were assessed by flow cytometry. The induction profile for all products tested including CRP 2.1 showed a 3 to 6- fold induction of the Ddit3 (reporter for protein damage) reporter gene in the absence of S9. However, there was no reporter gene induction in the presence of metabolic activation. None of the concentrations tested had an impact on cellular cytotoxicity indicating that the doses tested were not toxic, and the concentration range could be increased.

This preliminary work suggests that under the tested experimental conditions, ToxTracker is capable of distinguishing differences between flavors.

Ed Carmines, Manoj Misra, Sean Oh, Giel Hendrik, Bonnie Coffa, Katarina Aleksa

Affiliations: Chemular, Inc, Hudson, MI,USA, Labstat International, Kitchener, ON, CN, Toxys Inc., Leiden, The Netherlands