Oral nicotine pouch (NP) products may encourage adult tobacco consumers to switch from cigarettes to potentially reduced-harm smoke-free products. These products typically consist of nicotine and flavor ingredients. While many flavor ingredients have been well studied, the data from individual ingredients could inform the toxicological profile of the product. In this feasibility study, we evaluated flavored NPs that integrated new approach methodologies (NAMs) into a standard non-clinical test battery to better understand the mechanisms of any toxicity observed and to pave the way for animal-free testing strategies that can be used for similar products.

The genotoxic potential of NPs was evaluated in the in vitro micronuclei (MN) assay and the positive response indicated a potential hazard. Based on individual ingredient assessments and supported by in vitro testing, we identified maltol and ethyl maltol (“maltols”) as the driver of bioactivity in the in vitro MN assay for the whole product. Maltols, “Generally Recognized As Safe (GRAS)” for use in foods were reported to induce in vitro genotoxicity, but do not lead to in vivo sequelae. Studies also suggest that the in vivo (mouse) genotoxicity of maltols is related to the route of administration. Therefore, we hypothesized and investigated a dosimetry-based approach to interpret genotoxicity and carcinogenicity outcomes (in vitro versus in vivo) for maltols. Using open-source PBPK models, we also estimated the Cmax in the target tissue (e.g., plasma) under in vivo (rodent) exposure conditions and compared the levels to the in vitro concentrations. We also evaluated the NP in a combined in vivo MN and comet assay in rats following the ICH S2(R1) guidance and the negative in vivo results which also supported the dosimetry modeling. In summary, integrated approaches could enable a holistic evaluation of the genotoxic potential of an oral NP, building a case for NAM-based toxicological assessments without the need for confirmatory in vivo testing.