Establishing an ENDS In Vitro and In Vivo Ingredient Assessment Strategy Using a Toolbox Approach

Electronic Nicotine Delivery Systems (ENDS) comprise a wide variety of electronically powered devices used to heat an e-liquid that is typically composed of various flavors, with or without nicotine, diluted in propylene glycol- and vegetable glycerol-based solutions. While many flavor ingredients used in e-vapor products are “Generally Recognized as Safe” (GRAS) for oral consumption according to the Flavor and Extract Manufacturers Association (FEMA), many have insufficient safety data on inhalation exposures. Establishing a safety assessment strategy to separately analyze flavoring compounds in a comprehensive hazard characterization battery and in vitro and in vivo safety tests would be very challenging. More pragmatic approaches are needed for the combined evaluation of their potential toxicity when inhaled.

We conducted several non-clinical studies evaluating flavoring ingredients (>200), e-liquid formulations, and ENDS aerosols. The ingredients were initially screened for quality, purity, and FEMA GRAS status, followed by a comprehensive review of the available toxicological data. The flavor ingredients were assigned to 38 groups based on their chemical structure. Each group’s representative chemical was selected based on the specific toxicological properties of these compounds. The selected representatives were combined into a mixture that was used in traditional regulatory toxicology tests, alternative in vitro assays (e.g., high-content screening and ToxTracker), and chronic inhalation toxicology studies in A/J mice.

This flavor “toolbox” will help identify potential hazards associated with the tested flavorings and their respective chemical groups, thus expanding the relevance of a single animal study to more than 200 compounds, in line with 3R principles.