Nicotine pouches are a rapidly growing new smokeless tobacco product category introduced to the US market in 2019 by several tobacco companies. Nicotine pouches contain a white powder, claimed to be tobacco-free, consisting of filler, tobacco-sourced nicotine or nicotine salts, sweeteners and a variety of flavors. These include mint, menthol, wintergreen and sweet-associated flavors (fruity and candy flavors such as berry, citrus, cinnamon, coffee), marketed in color patterns resembling advertising for popular electronic cigarette brands, raising concerns that children and adolescents might be drawn to this new product category. Nicotine pouches are marketed with claims of reduced risk and enable consumers to discretely consume nicotine. Currently, only limited data are available about their exact chemical composition, their behavioral and addictive effects or toxicological properties. In this proposal, we hypothesize that: (i) product design and formulation of this novel smokeless tobacco product category enables efficient and fast release of nicotine, flavorants and sweeteners; (ii) flavorants released from nicotine pouches have adverse oral health effects; and (iii) flavorants and sweeteners in nicotine pouches facilitate and increase product use initiation. These hypotheses are based on our published work and preliminary data demonstrating that nicotine pouch products contain significant amounts of synthetic high-intensity sweeteners. These sweeteners facilitated consumption of nicotine pouch extracts in naïve mice. The extracts proved cytotoxic and compromised mitochondrial function in an oral epithelial cell line model, suggesting metabolic toxicity. The following Specific Aims are pursued: Aim 1: Analyze chemical composition of nicotine pouch products and evaluate nicotine and flavorant release. Aim 2: Examine the toxicological effects of nicotine pouch extracts on oral epithelial cells. Aim 3: Examine the effects of synthetic sweeteners and flavors in nicotine pouches on preference behavior in mice This proposal addresses key domains critical to the mission of the FDA CTP, including chemistry and Engineering, Toxicity, Behavior and Health Effects (Effects of product design characteristics, sweeteners, flavors and flavor changes on initiation, nicotine consumption, and oral health). If our hypotheses are confirmed the project will provide a rationale for FDA to regulate levels of sweeteners, flavors and other constituents in nicotine pouch products, thereby reducing the public health impact of this novel tobacco product category.