The oral tobacco-derived nicotine (OTDN) product category is one of fastest growing tobacco product segments in the United States offering alternative nicotine products that do not contain tobacco. A rigorous, nonclinical, toxicological assessment, including in vitro assays, is an integral part of a scientific evaluation of the potential health risks of OTDN products compared to other tobacco products. The toxicity assessment was performed using the standard regulatory in vitro assay battery: neutral red uptake (NRU) for cytotoxicity, Ames assay for mutagenicity, and micronuclei (MN) assay for genotoxicity. Ethanol extracts from a commercial OTDN product were first evaluated in comparison to condensate from reference cigarette (3R4F), extracts from reference moist smokeless tobacco (CRP2.1), and reference snus (CRP1.1). The 3R4F condensate was cytotoxic (IC₅₀: 3.67 µg/mL nicotine), mutagenic (strain TA98+S9 and TA1537+S9), and genotoxic (4h+S9) when tested up to 13.5 µg/mL nicotine. Extract from CRP 2.1 was not cytotoxic, weakly mutagenic (strain TA1537+S9), and non-genotoxic, whereas CRP 1.1 and OTDN extracts did not demonstrate cytotoxicity, mutagenicity or genotoxicity at the concentrations tested (up to 7-14 µg/mL nicotine). Two commercial OTDN products, CRP 1.1 and CRP 2.1, were also extracted using artificial saliva and subjected to in vitro testing at concentrations up to 162.5-367.5 µg/mL nicotine. The oral product extracts were found to be noncytotoxic, non-mutagenic, and non-genotoxic at the concentrations tested, except for CRP 2.1, which was positive in the MN assay (27h-S9). The results demonstrated that OTDN products had substantially lower toxicity than cigarettes, thus supporting their reduced-risk potential upon complete switching from smoking cigarettes.