Preclinical Assessment of Tobacco-Free Nicotine Pouches Demonstrates Reduced in Vitro Toxicity Compared with Tobacco Snus and Combustible Cigarette Smoke

Introduction: Tobacco harm reduction (THR) represents one of the most promising public health policies with continued product innovations crucial to making THR a reality. Tobacco-free nicotine pouches (TFNPs) are a recent THR product innovation available in a growing number of countries as a potentially less harmful alternative to traditional tobacco products. Materials and Methods: In this study, we compared the in vitro biological activity of two commercially available TFNPs, one snus product, and combustible cigarette smoke in three in vitro toxicological assays (neutral red uptake [NRU], in vitro micronucleus [IVM], and Ames test). An extraction method for TFNPs and snus in phosphate-buffered saline based on the International Organization of Standardization 10993-12 standard to assess the in vitro biological activity is also presented. Results and Discussion: Combustible cigarette smoke total particle matter (TPM) induced a statistically significant positive response in all three in vitro assays. By contrast, the TFNPs and snus extracts were negative in both the Ames and IVM assays, and demonstrated weak cytotoxicity in the NRU assay compared with TPM, under the conditions of test. When using the HepG2 cell line in the NRU assay, it was possible to differentiate between TFNP and snus extracts, with only snus extracts resulting in a measurable EC50 response. Conclusion: This initial preclinical in vitro toxicity assessment of TFNPs compared with both tobacco snus and combustible cigarette smoke indicates that the tested TFNPs have a substantially reduced in vitro toxicity activity compared with traditional tobacco products. Given this initial data, both further scientific exploration and public health discussion on these products are warranted.