In May 2016, the U.S. Food and Drug Administration Center for Tobacco Products (CTP) issued draft guidance for Premarket Tobacco Product Applications (PMTA) for Electronic Nicotine Delivery Systems (ENDS), which recommends inclusion of in vitro toxicology testing (e.g., genotoxicity and cytotoxicity studies). Specific to genotoxic potential, the draft guidance suggests using assays/methods outlined in International Conference on Harmonization (ICH) S2(R1) and Organization for Economic Cooperation and Development (OECD) guidelines. The draft guidance also recommends testing multiple test article concentrations in each assay, and using comparator products for hazard identification. We present an example of in vitro toxicology testing to support an ENDS PMTA. Bacterial mutagenicity (Ames test), in vitro micronucleus and cytotoxicity assays were conducted on ENDS e-liquids, ENDS product aerosols and U.S. market comparators in general accordance with applicable guidelines (ICH S2(R1); OECD 487 and 471; NIH Publication 07-4519 [2006] and Health Canada Official methods (T-501/T-502/T-503). Aerosol total particulate matter (TPM) and gas vapor phase (GVP) from ENDS products, and a combustible comparator were generated under intense puffing/smoking regimens for testing. Neat e-liquid samples were diluted and applied to the test systems. Test samples were evaluated up to concentrations resulting in toxicity or up to limits inherent in the assays. The ENDS e-liquids, TPM and GVP were either negative or demonstrated limited response at the highest concentrations tested in each of the assays, indicating a lack of toxicity and genotoxicity of the ENDS products under the experimental conditions tested. In contrast, with the exception of GVP in the Ames assay, the combustible market cigarette comparator was consistently positive, confirming that the assays conducted are sensitive to potential toxicants/genotoxicants found in combustible tobacco product emissions.