JUUL ENDS is a temperature-regulated product which was designed to minimize the generation of combustion related degradation byproducts in the aerosol. The overall purpose of this study was to test the cytotoxicity of the CHO-K1 cells in response to 24h exposure to various concentrations of ENDS aerosol using the NRU in vitro cytotoxicity assay using CHO-K1 cells according to OECD guidelines (TG 129). Methods: CH0-K1 cells were treated with ENDS aerosol, positive control, or vehicle control. JUUL ENDS aerosol was tested for three different flavor profiles. The cells were exposed to the different test articles; ENDS aerosol at the concentration of (0- 300 µg/mL) and compared with the mainstream smoke component (TPM) from 3R4F, at dose range of (0-150 µg/mL). Additionally, the cells were exposed to the positive control, SLS at concentrations 40 and 80 µg/mL. Results: Amongst all of the different samples tested, no aerosol mediated significant toxicity was observed at any of the concentrations tested for the test articles, Classic Menthol, Cool Mint, Cool Cucumber, as well as for PG/VG/Benzoic Acid/Nicotine (Carrier Control). At the doses tested, EC50 for the e-cig aerosol and carrier control aerosol could not be calculated because cell viability was greater than 70% at all concentrations tested. Additionally, TPM from the 3R4F cigarette which showed the expected toxicity with a calculated EC50 of 59.46 µg/mL with r2 = 0.98. In summary, under the experimental conditions and based on the criteria for Evaluation of Cytotoxic Response (ISO 10993-5), the test articles Classic Menthol, Cool Mint, Cool Cucumber, as well as for PG/VG/Benzoic Acid/Nicotine (Carrier Control) are considered non-cytotoxic.