In Vitro Toxicological Assessment of a Range of Oral Nicotine Pouches

Introduction: Sweden has a long-standing tradition of snus use over cigarette smoking, providing decades of epidemiology data establishing Swedish snus as a reduced-risk product as compared with traditional cigarettes. Oral nicotine pouches (ONPs) represent a recent development in oral nicotine delivery, being snus-like in both form and usage, but in a tobacco-free format, combining an inert filler with nicotine and flavors. The ONP category is, however, diverse, encompassing a wide range of characteristics including nicotine loading, flavor complexity, base (salt-based, sweet, and hybrid), pouch size, and fill weight. Methods: In this study, we make an in vitro assessment of a range of ONPs, testing 11 ONPs against two snus products: General Classical Blend White Portion Large Snus (Swedish Match, commercial) and the Swedish-style snus reference product 1S4 Reference (Center for Tobacco Reference Products [CTRP], University of Kentucky). The 11 commercial ONPs, which constitute nine Velo ONPs and two alternative-brand products, were selected to represent a wide matrix of variables. Products were extracted in cell culture media and evaluated for cytotoxicity and genotoxic potential using Real-Time Cell Analysis (RTCA) and ToxTracker assays, respectively. Results: The findings demonstrate that the tested ONPs and the commercial comparator General Classic White Portion Large Snus have a lower toxicological profile compared to the reference Kentucky 1S4 Swedish Style Snus. The inclusion of the broad spectrum of flavors and base formulation allowed for a preliminary assessment of their impact on toxicological outcomes. Conclusion: Results suggest minimal contribution of flavor and base to cytotoxic or genotoxic effects. Additionally, this study identified that osmolality should be considered when assessing these products due to the potential sensitivities to osmotic stress observed in the 2D cell cultures for some extracts. These insights are essential for guiding and shaping future product development and toxicological read-across within the ONP category.