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Prior electronic cigarette (EC) topography data are based on two video analyses with limited parameters. Alternate methods for measuring topography are needed to understand EC use and nicotine intake. This study evaluated EC topography with a CReSS Pocket device and quantified nicotine intake. Validation tests on pressure drop, flow rate, and volume confirmed reliable performance of the CReSS Pocket device. Twenty participants used Blu Cigs and V2 Cigs for 10 minute intervals with a 10-15 minute break between brands. Brand order was reversed and repeated within 7 days Data were analyzed to determine puff duration, puff count, volume, flow rate, peak flow, and inter-puff interval. Nicotine intake was estimated from cartomizer fluid consumption and topography data. Nine patterns of EC use were identified. The average puff count and inter-puff interval were 32 puffs and 17.9 seconds. All participants, except one, took more than 20 puffs/10 minutes. The averages for puff duration (2.65 seconds/puff), volume/puff (51 ml/puff), total puff volume (1,579 ml), EC fluid consumption (79.6 mg), flow rate (20 ml/s), and peak flow rate (27 ml/s) were determined for 10-minute sessions. All parameters except total puff count were significantly different for Blu versus V2 EC. Total volume for Blu versus V2 was four-times higher than for conventional cigarettes. Average nicotine intake for Blu and V2 across both sessions was 1.2 ± 0.5 mg and 1.4 ± 0.7 mg, respectively, which is similar to conventional smokers. EC puffing topography was variable among participants in the study, but often similar within an individual between brands or days. Puff duration, inter-puff interval, and puff volume varied from conventional cigarette standards. Data on total puff volume and nicotine intake are consistent with compensatory usage of EC. These data can contribute to the development of a standard protocol for laboratory testing of EC products.

BackgroundAs thousands of electronic cigarette (e-cigarette) refill fluids continue to be formulated and distributed, there is a growing need to understand the cytotoxicity of the flavouring chemicals and solvents used in these products to ensure they are safe. The purpose of this study was to compare the cytotoxicity of e-cigarette refill fluids/solvents and their corresponding aerosols using in vitro cultured cells.MethodsE-cigarette refill fluids and do-it-yourself products were screened in liquid and aerosol form for cytotoxicity using the MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) assay. The sensitivity of human pulmonary fibroblasts, lung epithelial cells (A549) and human embryonic stem cells to liquids and aerosols was compared. Aerosols were produced using Johnson Creek’s Vea cartomizer style e-cigarette.ResultsA hierarchy of potency was established for the aerosolised products. Our data show that (1) e-cigarette aerosols can produce cytotoxic effects in cultured cells, (2) four patterns of cytotoxicity were found when comparing refill fluids and their corresponding aerosols, (3) fluids accurately predicted aerosol cytotoxicity 74% of the time, (4) stem cells were often more sensitive to aerosols than differentiated cells and (5) 91% of the aerosols made from refill fluids containing only glycerin were cytotoxic, even when produced at a low voltage.ConclusionsOur data show that various flavours/brands of e-cigarette refill fluids and their aerosols are cytotoxic and demonstrate the need for further evaluation of e-cigarette products to better understand their potential health effects.

Thousands of electronic cigarette refill fluids are commercially available. The concentrations of nicotine and the solvents, but not the flavor chemicals, are often disclosed on product labels. The purpose of this study was to identify and quantify flavor chemicals in 39 commercial refill fluids that were previously evaluated for toxicity. Twelve flavor chemicals were identified with concentrations ≥1 mg/ml: cinnamaldehyde, menthol, benzyl alcohol, vanillin, eugenol, p -anisaldehyde, ethyl cinnamate, maltol, ethyl maltol, triacetin, benzaldehyde, and menthone. Transfer of these flavor chemicals into aerosols made at 3V and 5V was efficient (mean transfer = 98%). We produced lab-made refill fluids containing authentic standards of each flavor chemical and analyzed the toxicity of their aerosols produced at 3V and 5V using a tank Box Mod device. Over 50% of the refill fluids in our sample contained high concentrations of flavor chemicals that transferred efficiently to aerosols at concentrations that produce cytotoxicity. When tested with two types of human lung cells, the aerosols made at 5V were generally more toxic than those made at 3V. These data will be valuable for consumers, physicians, public health officials, and regulatory agencies when discussing potential health concerns relating to flavor chemicals in electronic cigarette products.

ObjectiveThe aim of this study was to evaluate the distribution, concentration and toxicity of cinnamaldehyde in electronic cigarette (e-cigarette) refill fluids and aerosols.MethodsThe distribution and concentration of cinnamaldehyde were determined in 39 e-cigarette refill fluids plus 6 duplicates using gas chromatography and mass spectrometry (GC/MS). A cinnamaldehyde toxicity profile was established for embryonic and adult cells using a live cell imaging assay, immunocytochemistry, the comet assay and a recovery assay.ResultsTwenty of the 39 refill fluids contained cinnamaldehyde at concentrations that are cytotoxic to human embryonic and lung cells in the MTT assay. Cinnamon Ceylon aerosol produced in a cartomizer-style e-cigarette was cytotoxic. Cinnamon Ceylon aerosols and refill fluid aerosols (80% propylene glycol or cinnamaldehyde/propylene glycol) made using a tank/boxmod e-cigarette were more cytotoxic at 5 V than 3 V. Using GC/MS, aerosols produced at 5 V contained 10 additional peaks not present in aerosol generated at 3 V. One of these, 2,3-butandione (diacetyl), was confirmed with an authentic standard. Cinnamaldehyde depolymerised microtubules in human pulmonary fibroblasts. At concentrations that produced no effect in the MTT assay, cinnamaldehyde decreased growth, attachment and spreading; altered cell morphology and motility; increased DNA strand breaks; and increased cell death. At the MTT IC50 concentration, lung cells were unable to recover from cinnamaldehyde after 2 hours of treatment, whereas embryonic cells recovered after 8 hours.ConclusionsCinnamaldehyde-containing refill fluids and aerosols are cytotoxic, genotoxic and low concentrations adversely affect cell processes and survival. These data indicate that cinnamaldehyde in e-cigarette refill fluids/aerosols may impair homeostasis in the respiratory system.

Electronic cigarettes (EC) have become part of mainstream culture. In 2016, the FDA classified EC as tobacco products and deemed they should be regulated as such. EC produce an aerosol from fluids that expose users to flavor chemicals generally regarded as safe for ingestion. When first introduced, little was known about how consumers would use these products or how they would affect human health. This project sought to assess EC topography, adapt 96-well plate assays for use with human embryonic stem cells (hESC), evaluate the cytotoxicity of EC refill fluids and aerosols, and identify flavor chemicals that may cause harm. Topography varied among users, but was often consistent within an individual between brands or days. Moreover, nicotine consumption and total puff volume were consistent with compensatory usage. hESC serve as an important model for evaluating the effects of EC on prenatal development that would be relevant for pregnant women who use these products. hESC, which are typically difficult to work with in quantitative assays, were adapted and validated for use in 96-well plate assays. Refill fluids and aerosols were then screened for cytotoxicity. Flavors, brands and solvent mixtures ranged in cytotoxicity with glycerin based products and cinnamon-flavored fluids being the most potent. To identify the cause of cytotoxicity, flavor chemicals were identified in a convenience sample of products. Twelve dominant flavor chemicals were found at concentrations >1 mg/ml. Cinnamaldehyde (CAD) was identified as a dominant flavor chemical in cinnamon-flavored products, and cytotoxicity was correlated with the amount of CAD/product. In several products, CAD was found at extremely high concentrations in both refill fluids (>100 mg/ml) and their aerosols. CAD was further tested in cell-based assays, and the overall impact of CAD on respiratory cells was studied at the air-liquid interface in a 3D model using a proteomics pathway analysis. CAD was genotoxic, impacted the cytoskeleton, and affected growth, motility and apoptosis, and upregulated several cell stress-based pathways in the human bronchial epithelium. This project found that some flavor chemicals, such as CAD, are present in EC products at high concentrations that could be harmful to human health. EC products, including flavor chemicals, should be regulated to insure user safety.

Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease.