Non-clinical in vitro studies were conducted to investigate the characteristics of tobacco free nicotine pouches alongside a reference snus product and marketed snus product. The investigation included the evaluation of nicotine transfer to artificial saliva, Ames tests in five Salmonella typhimurium strains, micronucleus assay, and neutral red uptake (NRU) cytotoxicity assay. Results from in vitro nicotine transfer tests suggested that most of the nicotine was extracted after approximately 20 minutes. Additionally, results from the Ames, micronucleus and NRU cytotoxicity assays indicated that the tobacco free nicotine pouch products were non-mutagenic, non-genotoxic and non-cytotoxic. Tests to investigate the cytotoxicity in human gingival epithelial cell cultures were also conducted, the results similarly showed a non-cytotoxic response.