Continuing change in both electronic cigarette (EC) aerosolization device design and the chemistry of the e- liquids that are vaped has complicated regulation as well as promoted use prevalence at all ages. Our chemi- cal / toxicological studies have focused on the e-liquids. The major changes that have occurred during the last five years in e-liquid chemistry include the use of many emerging ingredients (EIs). These include: a) non- confectionary cooling agents (e.g., the Wilkinson Sword “WS” chemicals originally developed for use in shaving creams); b) acids to protonate free-base nicotine to allow high nicotine products that are not harsh upon inhala- tion (e.g., JUULTM); c) synthetic nicotine rather than tobacco nicotine (as inspired by manufacturer hopes of evading U.S. regulation as “tobacco products”), and 4) sweet and fruity flavor chemicals (as stealth ingredients in non-regulated “tobacco”-flavored products). Ingredient changes are occurring quickly and constantly. There is an urgent need for work that tracks these changes and determines their toxicological implications. In Aim 1, 600 commercial products will be analyzed in a broad market survey to determine: a) the flavor chemicals on our target analyte list (183 compounds, including four worrisome EI chemicals in the WS class) plus any EIs at >20 g/mL; b) both the S and R nicotine forms plus three other tobacco alkaloids (e.g., nornicotine, which is a precursor for the potent carcinogen NNN) as indicators of use of natural / synthetic nicotine; and c) acid addi- tives. The data will be organized within the UCR /PSU Electronic Cigarette Data Collection. In Aim 2, we will investigate the potency and biological effects of both pure EIs (e.g., the WS compounds and acids) as well as worrisome flavor mixtures (e.g., EIs in combination with flavor chemicals, solvents, and nicotine). This first tox- icology effort will be carried out without heating. Aerosols will be generated in a cloud chamber using fluids containing physiological saline plus an individual ingredient chemical or a chemical mixture of concern. Expo- sures will be at the air/liquid interface (ALI) using “3D EpiAirway” tissue. Toxic effects will be identified using proteomic tissue analyses and confirmed using relevant mode-of action assays (e.g., inflammatory and oxida- tive stress responses). Margin of exposure (MOE) analyses will be carried out for each chemical tested to flag health effects including cancer risk. For Aim 3, we will carry out the second toxicology effort, repeating the work of Aim 2 but with heating using relevant EC heat levels during aerosol production. Commercial e-liquid products will also be tested. All exposures will be done at the ALI using a Cultex system and 3D EpiAirway tissue. Overall, the study will reveal the toxicity implications of new ingredient chemicals in the highly variable / changing EC marketplace. The results will reveal if the EC levels of these chemicals are toxic in 3D cultures of...