The in vitro toxicology testing of snus presents differing challenges to those encountered when testing combusted tobacco products, principally as the material to be assessed is insoluble (but absorbent) plant matter. By applying certain modifications to ISO guidelines that exist (for the biological evaluation of insoluble medical devices), snus extracts can be prepared which are suitable for use in various in vitro genotoxicity and cytotoxicity assays. The objective of the experimentation was to establish an extraction methodology for snus samples, that would permit a robust safety assessment in the Ames, Mouse Lymphoma, in vitro Micronucleus and Neutral Red Uptake assays. Snus samples with varying nicotine and water contents were extracted for 24 hours at 37 °C in both water and dimethyl sulphoxide (DMSO), at varying concentrations ranging from 200 to 500 mg (equivalent) per mL. At 500 mg (equivalent) per mL, the snus absorbed up to 85% of the extraction vehicle, making extraction at any higher concentration impractical. By measuring the nicotine content of the snus extracts, the percentage recovery of nicotine from the snus could be determined. Nicotine content was measured as 82-86% for aqueous snus extracts at 200 mg (equivalent) per mL, and at 73% for extracts at 500 mg (equivalent) per mL. A 500 mg (equivalent) per mL extract was therefore considered to be the highest extraction concentration that could practicably be extracted from snus or moist snuff. Subsequent in vitro genotoxicity assays performed using snus extracts at this concentration resulted in biological dose limiting effects in some cases, and/or achieved (or exceeded) exposure concentrations required in the standard regulatory guidelines applicable for these assays, and was therefore acceptable for safety assessment.