The emerging concern about chemicals in electronic cigarettes, even those without nicotine, demands the development of advanced criteria for their exposure and risk assessment. This study aims to highlight the sensitivity of lung nuclear receptors (NRs) to electronic cigarette e-liquids, independent of nicotine presence, and the influence of the sex variable on these effects. Adult male and female C57BL/6J mice were exposed to electronic cigarettes with 0%, 3%, and 6% nicotine daily (70 mL, 3.3 s, 1 puff per min/30 min) for 14 days, using the inExpose full body chamber (SCIREQ). Following exposure, lung tissues were harvested, and RNA extracted. The expression of 84 NRs was determined using the RT2 profiler mRNA array (Qiagen). Results exhibit a high sensitivity to e-liquid exposure irrespective of the presence of nicotine, with differential expression of NRs, including one (females) and twenty-four (males) in 0% nicotine groups compared to non-exposed control mice. However, nicotine-dependent results were also significant with seven NRs (females), fifty-three NRs (males) in 3% and twenty-three NRs (female) twenty-nine NRs (male) in 6% nicotine groups, compared to 0% nicotine mice. Sex-specific changes were significant, but sex-related differences were not observed. The study provides a strong rationale for further investigation.

E-liquids contain combinations of chemicals, with many enhancing the sensory attractiveness of the product. Studies are needed to understand and characterize e-liquid ingredients, particularly flavorings, to inform future research and regulations of these products. We identified common flavor ingredients in a convenience sample of commercial e-liquids using gas chromatography-mass spectrometry. E-liquid flavors were categorized by flavor descriptors provided on the product packaging. A Flavor Ingredient Wheel was developed to link e-liquid flavor ingredients with flavor categories. An analysis of 109 samples identified 48 flavor ingredients. Consistency between the labeled flavor and ingredients used to produce such flavor was found. Our novel Flavor Ingredient Wheel organizes e-liquids by flavor and ingredients, enabling efficient analysis of the link between ingredients and their flavor profiles and allowing for quick assessment of an e-liquid ingredient\'s flavor profile. Investigating ingredient profiles and identifying and classifying commonly used chemicals in e-liquids may assist with future studies and improve the ability to regulate these products.

BACKGROUND: Part of the appeal of e-cigarettes lies in their available flavors. To achieve attractive flavors, e-liquids contain many different flavoring agents, which allow many flavoring combinations. To advance our knowledge of e-liquid flavors and compositions and to evaluate the effect of legislation, we determined whether there are ingredient combinations that are frequently used together.
METHODS: We used e-cigarette ingredient data from the European Common Entry Gate system (EU-CEG) as available on 31 December 2022.
RESULTS: In e-liquids, we found 214 ingredient pairs with a co-occurrence odds ratio greater than 10. Together, these consisted of 62 unique ingredients. Network analysis revealed that ingredients were grouped together based on their flavor and/or chemical structure. We identified two densely connected regions (clusters) in the network. One consisted of six ingredients with sweet-vanilla-creamy flavors. The second cluster consisted of 13 ingredients. While some of these have fruity flavors, others, such as alkyl carboxylic acids and dimethyl sulfide, are known to have unpleasant flavors. Additional data and literature analyses indicated that alkyl carboxylic acids can contribute to a creamy and sweet-fruity taste, whereas dimethyl sulfide can contribute to a more refined fruity taste.
CONCLUSIONS: These results exemplify that the flavor of e-liquids is not just the sum of its parts. Big data analyses on product data can be used to detect such patterns, but expert knowledge and additional data are needed for further interpretation. Monitoring of e-liquid flavors as well as ingredients will remain important to regulate e-liquid product attractiveness.

Scientific progress and ethical considerations are increasingly shifting the toxicological focus from in vivo animal models to in vitro studies utilizing physiologically relevant cell cultures. Consequently, we evaluated and validated a three-dimensional (3D) model of the human lung using Calu-3 cells cultured at an air-liquid interface (ALI) for 28 days. Assessment of seven essential genes of differentiation and transepithelial electrical resistance (TEER) measurements, in conjunction with mucin (MUC5AC) staining, validated the model. We observed a time-dependent increase in TEER, genetic markers of mucus-producing cells (muc5ac, muc5b), basal cells (trp63), ciliated cells (foxj1), and tight junctions (tjp1). A decrease in basal cell marker krt5 levels was observed. Subsequently, we utilized this validated ALI-cultured Calu-3 model to investigate the adversity of the aerosols generated from three flavored electronic cigarette (EC) e-liquids: cinnamon, vanilla tobacco, and hazelnut. These aerosols were compared against traditional cigarette smoke (3R4F) to assess their relative toxicity. The aerosols generated from PG/VG vehicle control, hazelnut and cinnamon e-liquids, but not vanilla tobacco, significantly decreased TEER and increased lactate dehydrogenase (LDH) release compared to the incubator and air-only controls. Compared to 3R4F, there were no significant differences in TEER or LDH with the tested flavored EC aerosols other than vanilla tobacco. This starkly contrasted our expectations, given the common perception of e-liquids as a safer alternative to cigarettes. Our study suggests that these results depend on flavor type. Therefore, we strongly advocate for further research, increased user awareness regarding flavors in ECs, and rigorous regulatory scrutiny to protect public health.

Electronic cigarettes (e-cigarettes, vaping products) have become increasingly popular, with recent increases in use associated with closed systems delivering higher concentrations of nicotine. Most vaping products designed as an alternative to combustible cigarettes contain nicotine. A number of published studies have examined the reported concentrations of nicotine in vaping liquids (e-liquids) and found discrepancies between labelled and measured levels. Some discrepancy can also be explained by the lack of stability of nicotine in these types of products. Recently, a chemical analysis method for the quantitative determination of low and high levels of nicotine in vaping liquids was developed. This method uses dilution with acetonitrile prior to analysis with gas chromatograph mass spectrometry (GC-MS) in single ion monitoring mode (SIM). The developed method was validated using a laboratory-prepared vaping liquid as well as commercially available, nicotine-free products fortified with nicotine in the laboratory. The method detection limit (MDL) and the limit of quantitation (LOQ) for nicotine were calculated to be 0.002 mg/mL and 0.006 mg/mL, respectively. The newly developed method was applied to quantify nicotine in commercially available vaping liquids of various flavour profiles and across a wide range of nicotine concentrations, including those with nicotine salts. Furthermore, a subset of vaping liquids were analyzed to elucidate nicotine stability in various product subtypes. After a period of six months of accelerated storage to mimic one year, the overall mean percent of the original nicotine concentration remaining in the salt-based vaping products was 85% (minimum 64%, maximum 99%) while in the free-base nicotine products it was 74% (minimum 31%, maximum 106%). Nicotine stability in vaping liquids was found to be influenced by the nicotine form (pH) of formulation and its chemical composition. Non-targeted, qualitative analysis of chemical composition of vaping products showed that most constituents were identified and found to be remaining in the products following stability trials; however, three new compounds were tentatively identified in some vaping liquids at the end of the stability trials. Stability studies and the accurate quantitation of nicotine in vaping products can help inform product standards related to the safety, quality and utility of vaping products as a smoking cessation tool.

Electronic cigarettes are generally recognized as a safer alternative than conventional cigarettes. Nevertheless, previous research suggests metal (loid) leaching due to coil contact, potentially transferring to the e-liquid and its aerosolized form. In this study, Cr, Cd, Ni, and Pb levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) on 17 samples of e-liquids with different chemical properties (e.g., pH, nicotine content, flavoring, free-base, and nicotine salts). Twelve e-liquids were then put in contact with 36-gauge Kanthal A-1, Nichrome 80, Stainless steel 317 L and disposable coils such as Juul, and Aspire BVC for three days at 200-250 °C for 1 h each day. Metal levels expressed as mean (standard deviation) metal concentration, were below detection (Cd) to very low in bottle samples (Ni ≤ 76 (18); Pb ≤ 16 (1.5); and Cr ≤ 386 (15.6) μg/kg). In the coil extracts, varying concentrations of the same metal (loid) were found, indicating that metal leaching capacity may differ per sample. All samples contained Ni and Cr, followed by Pb to a much lesser extent. Cd levels were mostly below detection limits. Coil + e-liquid combinations with the highest Ni, Cr, and Pb concentrations were: Aspire BVC + Melon 0 mg/mL: Ni = 1.22 E+04 (281); Aspire BVC + Hit Nicotine 40 mg/mL: Cr = 864 (116); and Nichrome 80 + Melon 0 mg/mL: Pb = 56 (5) μg/kg. Overall, results suggest that nicotine salts at 40 mg/mL enhance Cr and Ni transfer. Stainless steel 317 L released very low metal concentrations. A conservative screening level risk characterization showed that 10.5% and 3.5% of the coil extracts may exceed Ni and Cr (III) safe concentrations, respectively. In the aerosol phase, 8.8% of samples might be above Ni equivalent daily dose for chronic exposure and 1.8% for intermediate exposure. Further studies on coil metal leaching could aid in establishing coil manufacturing regulations.

BACKGROUND: Menthol is a characterizing flavor in combustible cigarettes and electronic nicotine delivery systems (ENDS). E-liquids are recognized as an important reason for ENDS use. Yet, compared to menthol cigarettes, menthol flavored e-liquids and other cooling flavors, including mint and \'ice\' for ENDS, are an understudied area of investigation. The present study examined cooling flavored e-liquid website marketing among brick-and-mortar vape shops in Greater Los Angeles Area.
METHODS: A total of 104 brick-and-mortar vape shops were identified, and 38 had active websites. Over a 30-day period (28 September - 28 October 2020), we collected marketing for all flavored refillable e-liquids (photos/images) from each website. Data were collected for 1330 products. Only refillable e-liquid bottles were selected for this study, excluding prefilled pod vapes and disposable vapes. Each e-liquid per website was coded (e.g. menthol or not) and grouped into categories (e.g. type of menthol). The three flavor descriptor categories that the e-liquids were grouped into were menthol, mint, or ice. An \'other\' category was also created for identified e-liquids with flavor descriptors that did not mention menthol, mint, or ice.
RESULTS: A total of 1330 e-liquid flavors were identified from 38 active websites. Among them, 219 were coded as menthol/mint/ice flavor e-liquids. Frequencies included ice/iced/icy (n=123; 56%), menthol (n=32; 15%), mint (n=23; 11%), and other (n=41; 19%). Of the 123 ice-flavor e-liquids, 70.3% (n=83) were in combination with fruit (e.g. \'Apple Ice\', \'Grape Iced\', \'Icy Mango\'). Of the 32 menthol-flavored e-liquids, 63.3% (n=19) were in combination with fruit (e.g. \'Dragon Fruit Menthol\', \'Blue Raspberry Menthol\', \'Fresh Peach Menthol\'). Flavors in the other category were ice-related flavor descriptors (e.g. \'Arctic Air\', \'Brain Freeze\', \'Frozen Hulk Tears\').
CONCLUSIONS: Brick-and-mortar vape shops in the Greater Los Angeles Area market a variety of cooling flavor e-liquids on their websites. Such marketing focused largely on ice fruit flavor combinations. Future research is needed to understand how exposure to and appeal of cooling flavored e-liquid marketing differs among diverse consumer groups, including adult menthol cigarette smokers and non-smoking youth.

BACKGROUND: Cigarette smoking poses many health risks and can cause chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer of the lung and other organs. Smokers can substantially reduce their risks of these diseases by quitting, but nicotine addiction makes this difficult. Alternatives, such as electronic cigarettes (e-cigarettes), may provide a similar dose of nicotine, but expose users to fewer toxic chemicals than traditional cigarettes and may still be harmful especially for dual users, therefore, we sought to develop bioassays that can assess the potential toxicity and inflammatory response induced by e-cigarette liquids (e-liquids) with and without flavors.
METHODS: E-liquids with varying nicotine content and flavors were aerosolized through growth media and exposed to human bronchial epithelial cell line (BEAS-2B) and human monocyte-macrophage cell line (THP-1) in vitro. Cytotoxicity in response to e-cigarette aerosols was measured by MTT assay in BEAS-2B cells and inflammatory response was measured by TNF-α, IL-6, IL-8, and MCP-1 released from THP-1 cells. In addition, the oxidative stress marker, REDD1, and impact on phagocytosis, was assessed following exposure of BEAS-2B and THP-1 derived macrophages, respectively. Cigarette smoke extract was used as a positive control with known cytotoxicity and impairment of inflammatory response.
RESULTS: E-cigarette aerosols induced moderate cellular toxicity in bronchial epithelial cells. Our data also show that low nicotine levels are less damaging to the bronchial epithelial cells, and flavors in e-liquids influence the combined inflammatory response markers, phagocytosis, and REDD1 when examined in vitro.
CONCLUSIONS: Our in vitro bioassays can be utilized to effectively measure flavor and nicotine-induced effects of e-cigarettes on combined inflammatory response and cytotoxicity in human macrophages and human bronchial epithelial cells, respectively.

It is currently understood that tobacco smoking is a major cause of pulmonary disease due to pulmonary/lung inflammation. However, due to a highly dynamic market place and an abundance of diverse products, less is known about the effects of e-cigarette (E-cig) use on the lung. In addition, varieties of E-cig liquids (e-liquids), which deliver nicotine and numerous flavor chemicals into the lungs, now number in the 1000s. Thus, a critical need exists for safety evaluations of these E-cig products. Herein, we employed a \"2-stage in vivo screening platform\" (zebrafish to mouse) to assess the safety profiles of e-liquids. Using the zebrafish, we collected embryo survival data after e-liquid exposure as well as neutrophil migration data, a key hallmark for a pro-inflammatory response. Our data indicate that certain e-liquids induce an inflammatory response in our zebrafish model and that e-liquid exposure alone results in pro-inflammatory lung responses in our C57BL/6J model, data collected from lung staining and ELISA analysis, respectively, in the mouse. Thus, our platform can be used as an initial assessment to ascertain the safety profiles of e-liquid using acute inflammatory responses (zebrafish, Stage 1) as our initial metric followed by chronic studies (C57BL/6J, Stage 2).

As of February 18, 2020, the e-cigarette, or vaping, product use associated lung injury (EVALI) outbreak caused the hospitalization of a total of 2,807 patients and claimed 68 lives in the United States. Though investigations have reported a strong association with vitamin E acetate (VEA), evidence from reported EVALI cases is not sufficient to rule out the contribution of other chemicals of concern, including chemicals in either THC or non-THC products. This study characterized chemicals evolved when diluent oils were heated to temperatures that mimic e-cigarette, or vaping, products (EVPs) to investigate production of potentially toxic chemicals that might have caused lung injury. VEA, vitamin E, coconut, and medium chain triglyceride (MCT) oil were each diluted with ethanol and then tested for constituents and impurities using a gas chromatograph mass spectrometer (GC/MS). Undiluted oils were heated at 25°C (control), 150°C, and 250°C in an inert chamber to mimic a range of temperatures indicative of aerosolization from EVPs. Volatilized chemicals were collected using thermal desorption tubes, analyzed using a GC/MS, and identified. Presence of identified chemicals was confirmed using retention time and ion spectra matching with analytic standards. Direct analysis of oils, as received, revealed that VEA and vitamin E were the main constituents of their oils, and coconut and MCT oils were nearly identical having two main constituents: glycerol tricaprylate and 2-(decanoyloxy) propane-1,3-diyl dioctanoate. More chemicals were measured and with greater intensities when diluent oils were heated at 250°C compared to 150°C and 25°C. Vitamin E and coconut/MCT oils produced different chemical emissions. The presence of some identified chemicals is of potential health consequence because many are known respiratory irritants and acute respiratory toxins. Exposure to a mixture of hazardous chemicals may be relevant to the development or exacerbation of EVALI, especially when in concert with physical damage caused by lung deposition of aerosols produced by aerosolizing diluent oils.