Nicotine, Humectants, and Tobacco-Specific Nitrosamines (TSNAs) in IQOS Heated Tobacco Products (HTPs): A Cross-Country Study.

Heated Tobacco Products (HTPs) purport to reduce exposure to tobacco-related toxicants compared to combustible cigarettes. This cross-sectional study examined the content of nicotine, two humectants (propylene glycol (PG) and vegetable glycerin (VG)), and four tobacco-specific nitrosamines (TSNAs: NNN, NNK, NAT, and NAB) in the tobacco filler of a popular HTP brand (IQOS). Non-menthol and menthol IQOS sticks were purchased from nine countries between 2017 and 2020 and were classified into two versions ("Bold" and "Light") using Philip Morris's flavor descriptors. The average nicotine concentration was 4.7 ± 0.5 mg/stick, and the highest nicotine concentration was found in products from Japan (5.1 ± 0.2 mg/stick). VG was the dominant humectant found in all sticks, with an average concentration of (31.5 ± 2.3 mg/stick). NNN, NNK, and NAT were substantially higher in the "Bold" sticks than the "Light" sticks. Significant differences between countries for TSNAs were also observed: the NAT and NAB contents were the highest in the "Light" products from Canada (192.5 ± 24.1 and 22.9 ± 1.0 ng/stick, respectively); the NNK concentration was the highest in the "Bold" products from Poland (64.8 ± 7.9 ng/stick); and the highest NNN concentrations were observed in the "Bold" products from South Africa (488.9 ± 26.7 ng/stick). As NNN and NNK are known human carcinogens, and as humectants like PG and VG can degrade into toxic carbonyl compounds upon heating, monitoring the concentration of these chemicals in HTPs is important for protecting users' health and ensuring compliance with regulations.

Do alternative tobacco products induce less adverse respiratory risk than cigarettes?

RATIONALE: Due to the relatively short existence of alternative tobacco products, gaps exist in our current understanding of their long-term respiratory health effects. We therefore undertook the first-ever side-by-side comparison of the impact of chronic inhalation of aerosols emitted from electronic cigarettes (EC) and heated tobacco products (HTP), and combustible cigarettes (CC) smoke. OBJECTIVES: To evaluate the potential differential effects of alternative tobacco products on lung inflammatory responses and efficacy of vaccination in comparison to CC. METHODS: Mice were exposed to emissions from EC, HTP, CC, or air for 8 weeks. BAL and lung tissue were analyzed for markers of inflammation, lung damage, and oxidative stress. Another group was exposed for 12 weeks and vaccinated and challenged with a bacterial respiratory infection. Antibody titers in BAL and sera and pulmonary bacterial clearance were assessed. MAIN RESULTS: EC- and HTP-aerosols significantly augmented lung immune cell infiltrates equivalent to that achieved following CC-exposure. HTP and CC significantly increased neutrophil numbers compared to EC. All products augmented numbers of B cells, T cells, and pro-inflammatory IL17A+ T cells in the lungs. Decreased lung antioxidant activity and lung epithelial and endothelial damage was induced by all products. EC and HTP differentially augmented inflammatory cytokines/chemokines in the BAL. Generation of immunity following vaccination was impaired by EC and HTP but to a lesser extent than CC, with a CC > HTP > EC hierarchy of suppression of pulmonary bacterial clearance. CONCLUSIONS: HTP and EC-aerosols induced a proinflammatory pulmonary microenvironment, lung damage, and suppressed efficacy of vaccination.

Still 'Cool': tobacco industry responds to state-wide menthol ban with synthetic coolants.

INTRODUCTION: In December 2022, California (CA) enforced a voter-approved regulation restricting the retail sale of flavoured tobacco products, including menthol cigarettes. Shortly after, new products emerged on the market containing similar blue and green package colours yet with 'non-menthol' descriptors. Using chemical analyses, we measured the content of menthol and 15 other cooling chemicals in Californian cigarettes with 'non-menthol' descriptors and compared concentrations to similar 'menthol'-labelled counterparts available in New York State (NY). METHODS: A convenience sample of 10 brands and types of cigarettes in CA were purchased based on package colours suggesting a cooling effect and/or 'non-menthol' descriptors. The exact brand and type of cigarettes (with menthol descriptors) were purchased in NY. Cigarettes from CA were compared with equivalent cigarettes from NY on package design and colours, cigarette physical characteristics and the presence of cooling additives. RESULTS: Menthol was not detected in any CA cigarette, except for Maverick-green box type, while its presence was confirmed in most NY counterpart products. A synthetic cooling chemical WS-3 was not detected in any NY cigarettes but was detected in four CA brands and types with implied cooling effect, ranging from 1.24±0.04 to 1.97±0.05 mg/cigarette. CONCLUSION: While manufacturers have removed menthol descriptors from CA packaging and the menthol ingredient from cigarettes, synthetic cooling chemicals detected in several CA brands suggest that cooling sensory effects may still be sustained. Policymakers must consider both the chemical ingredients themselves and sensory effects in future regulatory approaches.

Heavy metals in ENDS: a comparison of open versus closed systems purchased from the USA, England, Canada and Australia.

INTRODUCTION: Electronic nicotine delivery systems (ENDS) are known to contain heavy metals such as lead (Pb), nickel (Ni) and chromium (Cr). The presence of heavy metals in ENDS may be due to contamination of e-liquids or leaching from elements of the ENDS device. This study investigates differences in ENDS metal concentrations between product type, year of purchase, country of purchase and e-liquid flavour. METHODS: Various open-system (refill e-liquids; n=116) and closed-system (prefilled with e-liquid; n=120) products were purchased in 2017 and 2018 from the USA, England, Canada and Australia. Electrothermal atomic absorption spectroscopy was used to analyse each product for Pb, Ni and Cr. Multiple linear regression and Kruskal-Wallis non-parametric statistical tests were conducted using GraphPad. RESULTS: Linear regression showed system type, year of purchase (not supported by Kruskal-Wallis), country of purchase and flavour type each had significant impacts on heavy metal concentrations. Open-system e-liquid samples showed no quantifiable levels of heavy metals. Closed-system samples contained concerningly high concentrations of Pb, Ni and Cr. Closed-system samples from the USA commonly displayed higher average heavy metal concentrations than those from England. Some fruit and mint-flavoured closed-system products showed higher heavy metal concentrations than tobacco-flavoured products. CONCLUSION: The presence of heavy metals only in closed-system products suggests that metals may be leaching from ENDS device parts. Highly variable heavy metal concentrations between ENDS products demonstrate that various product characteristics may affect the degree of leaching and that there is a need for further regulation of these products.

Characterisation of vaping liquids used in vaping devices across four countries: results from an analysis of selected vaping liquids reported by users in the 2016 ITC Four Country Smoking and Vaping Survey.

OBJECTIVES: This study presents an analysis of vaping products (VPs) purchased in the USA, Canada, England and Australia and assesses whether differences in regulations were associated with differences in the chemical composition of the VPs. METHODS: April-September 2017, a total of 234 VP refill liquids and prefilled cartridges were purchased in convenience samples of retail locations in each country. Products were chosen from brands and styles most commonly reported by current VP users in the 2016 ITC Four Country Smoking and Vaping Survey. All products were tested for nicotine, tobacco-specific nitrosamines (TSNAs), minor tobacco alkaloids, organic acids and flavouring chemicals. RESULTS: Consistent with the laws in Canada and Australia at the time of product purchase, nicotine was not detected in any of the VPs (n=10 in Canada; n=15 in Australia). US liquids (n=54) had a mean nicotine concentration of 16.2 mg/mL, (range=0.0-58.6) and English liquids (n=166) had a mean concentration of 11.9 mg/mL ((range=0.0-31.2) F(3244)=12.32, p<0.001). About 5% of English samples exceeded the UK's 20 mg/mL nicotine limit. Substantial country differences were observed in levels ofTSNAs, with the USA being higher than elsewhere. Of all products tested, 18.8% contained at least one organic acid. Liquids purchased in England contained far more identifiable flavouring chemicals than those in the other countries. CONCLUSIONS: VP composition, particularly with respect to nicotine and flavouring, varies by country, likely reflecting both marketplace preferences and country-specific regulations. There are differences between nicotine levels claimed on the package and actual levels, particularly in England.

Comparing POD and MOD ENDS Users' Product Characteristics, Use Behaviors, and Nicotine Exposure.

INTRODUCTION: POD electronic nicotine delivery systems (ENDS), often containing high concentrations of nicotine salts, have replaced MODs (ie, open/modifiable devices) as the most popular devices. The purpose of this study was to compare device/liquid characteristics, use behavior, and nicotine exposure between POD and MOD users. METHODS: Data from the initial visit of a prospective observational study of exclusive ENDS users compared MOD (n = 48) and POD (n = 37) users. Participants completed questionnaires on demographic characteristics, patterns of ENDS use, and ENDS features. A urine sample was collected to test for cotinine and an ENDS liquid sample was collected to test for nicotine and salts. Puff topography was captured during an ad libitum bout at the end of the session. RESULTS: MOD and POD users did not differ on demographic characteristics. MOD users reported purchasing more liquid in the past month than POD users (180.4 ±â€…28.0 vs. 50.9 ±â€…9.0 ml, p < .001). Differences in characteristics of devices used by MOD and POD users included flavor type (p = .029), nicotine concentration (liquids used by MOD users contained less nicotine than those used by POD users: 8.9 ±â€…2.0 vs. 41.6 ±â€…3.2 mg/ml, p < .001), and presence of the nicotine salt (fewer MOD liquids had salts present than POD liquids: 11.9% vs. 77.4%, p < .001). User groups did not differ on urinary cotinine levels or puff topography (ps > .05). CONCLUSIONS: Despite different characteristics of MOD and POD ENDS, users of those products are exposed to similar amounts of nicotine, likely due to using more liquid among MOD users. IMPLICATIONS: This study directly compares ENDS product characteristics, user behavior, and nicotine exposure between MOD and POD ENDS users. Although POD products contained higher nicotine concentrations compared to MOD products, users of PODs reported consuming less liquid than MOD users. Ultimately, MOD and POD users were exposed to similar levels of nicotine, suggesting users behaviorally compensate for differences in product characteristics.

The E-cigarette or Vaping Product Use-Associated Lung Injury Epidemic: Pathogenesis, Management, and Future Directions: An Official American Thoracic Society Workshop Report.

E-cigarette or vaping product use-associated lung injury (EVALI) is a severe pulmonary illness associated with the use of e-cigarettes or vaping products that was officially identified and named in 2019. This American Thoracic Society workshop was convened in 2021 to identify and prioritize research and regulatory needs to adequately respond to the EVALI outbreak and to prevent similar instances of disease associated with e-cigarette or vaping product use. An interdisciplinary group of 26 experts in adult and pediatric clinical care, public health, regulatory oversight, and toxicology were convened for the workshop. Four major topics were examined: 1) the public health and regulatory response to EVALI; 2) EVALI clinical care; 3) mechanisms contributing to EVALI; and 4) needed actions to address the health effects of EVALI. Oral presentations and group discussion were the primary modes used to identify top priorities for addressing EVALI. Initiatives including a national EVALI case registry and biorepository, integrated electronic medical record coding system, U.S. Food and Drug Administration regulation and enforcement of nicotine e-cigarette standards, regulatory authority over nontobacco-derived e-cigarettes, training in evaluating exogenous exposures, prospective clinical studies, standardized clinical follow-up assessments, ability to more readily study effects of cannabinoid e-cigarettes, and research to identify biomarkers of exposure and disease were identified as critical needs. These initiatives will require substantial federal investment as well as changes to regulatory policy. Overall, the workshop identified the need to address the root causes of EVALI to prevent future outbreaks. An integrated approach from multiple perspectives is required, including public health; clinical, basic, and translational research; regulators; and users of e-cigarettes. Improving the public health response to reduce the risk of another substantial disease-inducing event depends on coordinated actions to better understand the inhalational toxicity of these products, informing the public of the risks, and developing and enforcing regulatory standards for all e-cigarettes.

Changes in product labelling practices and the use of flavouring chemical additives in vaping products after enactment of statewide flavour legislation.

INTRODUCTION: On 18 May 2020, New York State enacted legislation banning the sale of vaping products with distinguishable flavours (other than tobacco). According to this new statute, vaping products are deemed flavoured if they include a statement, whether expressed or implied, that have distinguishable tastes or aromas other than tobacco. This study aimed to determine how manufacturers responded. METHODS: We collected 555 vaping products from daily vapers (238 preban and 317 postban). We compared preban and postban labelling of products for expressed and implied flavour descriptions, graphics and colours. Flavouring chemicals and concentrations were identified using chromatography methods and were compared preban and postban. RESULTS: Analysis of the labels preban and postban did not reveal a change in products with expressed flavoured descriptors (45.8% vs 44.2%) and a minimal decrease in implied descriptors (22.3% vs 14.5%). An increase in products without any descriptors was observed (28.2% vs 37.2%) notably within products from a popular pod brand. The average concentration of eight popular flavourings identified preban was 1.4±2.7 compared with 2.3±3.5 mg/mL (p<0.001) postban. No significant changes between individual flavouring concentrations in the most popular refill solutions and pods were found. CONCLUSION: While a majority of products appeared to remain non-compliant, this study suggests that enactment of legislation on vaping products making expressed or implied flavour claims may result in some manufacturer changes to product labelling including removal of flavour descriptors. However, use of flavouring additives in vaping products appeared not to be impacted by the ban.

Removal of mango-flavoured Juul pods created opportunity for adulterated mango Juul-compatible pods with altered chemical constituents.

INTRODUCTION: Juul is a leading electronic cigarette (e-cigarette) brand in the USA. By November 2019, Juul pre-emptively limited online and in-store sales of non-tobacco or menthol-flavoured pods ahead of impending flavour bans. Since this removal, sale of mango-flavoured Juul-compatible pods was introduced to the market by smaller companies. The aim of this study was to compare chemical constituents of original Juul mango pods with mango-flavoured Juul-compatible pods. METHODS: Juul and 16 brands of Juul-compatible mango-flavoured pods were purchased online in May 2018 (original Juul) and November 2019 (Juul-compatible), after Juul voluntarily removed their flavoured pods from the market. Liquid was extracted from pods and analysed using chromatography and mass spectrometry methods for nicotine concentration, solvent ratios, nicotine salt identification, as well as flavouring identification and quantitation. RESULTS: Juul-compatible pods had a significantly lower average nicotine concentration compared with original Juul pod (42.8±8.9 vs 57.2±0.9 mg/mL, p<0.0001). Nicotine benzoate was used in original Juul pod and all Juul-compatible pods. The propylene glycol to vegetable glycerin volumetric ratio of Juul-compatible pods averaged 55:45, while the original Juul pod was 35:65 (p<0.0001). Total number of flavouring chemicals detected was significantly higher in Juul-compatible pods as compared with Juul (p<0.0001). In Juul-compatible pods, average concentrations of benzyl alcohol (fruity flavouring) were 0.8±1.3 mg/mL, approximately 27 times higher than in original Juul pod (p<0.0001). CONCLUSIONS: Adulterated Juul-compatible products may expose e-cigarette consumers to more chemical constituents at higher concentrations than previously found in the original product, despite similarity in product design.

Genetic structure of dinoflagellate symbionts in coral recruits differs from that of parental or local adults.

The symbiotic relationship between dinoflagellate algae in the family Symbiodiniaceae and scleractinian corals forms the base of the tropical reef ecosystem. In scleractinian corals, recruits acquire symbionts either "vertically" from the maternal colony or initially lack symbionts and acquire them "horizontally" from the environment. Regardless of the mode of acquisition, coral species and individual colonies harbor only a subset of the highly diverse complex of species/taxa within the Symbiodiniaceae. This suggests a genetic basis for specificity, but local environmental conditions and/or symbiont availability may also play a role in determining which symbionts within the Symbiodiniaceae are initially taken up by the host. To address the relative importance of genetic and environmental drivers of symbiont uptake/establishment, we examined the acquisition of these dinoflagellate symbionts in one to three-month-old recruits of Orbicella faveolata to compare symbiont types present in recruits to those of parental populations versus co-occurring adults in their destination reef. Variation in chloroplast 23S ribosomal DNA and in three polymorphic microsatellite loci was examined. We found that, in general, symbiont communities within adult colonies differed between reefs, suggesting that endemism is common among symbiont populations of O. faveolata on a local scale. Among recruits, initial symbiont acquisition was selective. O. faveolata recruits only acquired a subset of locally available symbionts, and these generally did not reflect symbiont populations in adults at either the parental or the outplant reef. Instead, symbiont communities within new recruits at a given outplant site and region tended to be similar to each other, regardless of parental source population. These results suggest temporal variation in the local symbiont source pool, although other possible drivers behind the distinct difference between symbionts within O. faveolata adults and new generations of recruits may include different ontogenetic requirements and/or reduced host selectivity in early ontogeny.

Acute Effects of Heated Tobacco Product (IQOS) Aerosol Inhalation on Lung Tissue Damage and Inflammatory Changes in the Lungs.

INTRODUCTION: Emerging heated tobacco products (HTPs) were designed to reduce exposure to toxicants from cigarette smoke (CS) by avoiding burning tobacco and instead heating tobacco. We studied the effects of short-term inhalation of aerosols emitted from HTP called IQOS, on lung damage and immune-cell recruitment to the lungs in mice. METHODS: Numerous markers of lung damage and inflammation including albumin and lung immune-cell infiltrates, proinflammatory cytokines, and chemokines were quantified in lungs and bronchoalveolar (BAL) fluid from IQOS, CS, or air-exposed (negative control) mice. RESULTS: Importantly, as a surrogate marker of lung epithelial-cell damage, we detected significantly increased levels of albumin in the BAL fluid of both HTP- and CS-exposed mice compared with negative controls. Total numbers of leukocytes infiltrating the lungs were equivalent following both IQOS aerosols and CS inhalation and significantly increased compared with air-exposed controls. We also observed significantly increased numbers of CD4+IL-17A+ T cells, a marker of a T-cell immune response, in both groups compared with air controls; however, numbers were the highest following CS exposure. Finally, the numbers of CD4+RORγt+ T cells, an inflammatory T-cell subtype expressing the transcription factor that is essential for promoting differentiation into proinflammatory Th17 cells, were significantly augmented in both groups compared with air-exposed controls. Levels of several cytokines in BAL were significantly elevated, reflecting a proinflammatory milieu. CONCLUSIONS: Our study demonstrates that short-term inhalation of aerosols from IQOS generates damage and proinflammatory changes in the lung that are substantially similar to that elicited by CS exposure. IMPLICATIONS: Exposure of mice to IQOS, one of the candidate modified-risk tobacco products, induces inflammatory immune-cell accumulation in the lungs and augments the levels of proinflammatory cytokines and chemokines in the BAL fluid. Such an exacerbated pulmonary proinflammatory microenvironment is associated with lung epithelial-cell damage in IQOS-exposed mice, suggesting a potential association with the impairment of lung function.

Acute Effect of Electronic Cigarette-Generated Aerosol From Flavored CBD-Containing Refill Solutions on Human Bronchial Epithelial Cells.

Introduction: Although electronic cigarettes (e-cigarettes) were originally developed to deliver aerosolized nicotine to lungs, recent data have shown that consumers also use them for inhalation of other drugs, including cannabidiol (CBD). The aim of this study was to test the acute inhalation toxicity of flavored CBD-containing aerosols emitted from e-cigarettes. Methods: Bronchial epithelial cells (H292) cells were exposed to aerosol generated from e-cigarettes refilled either with (1) propylene glycol solvent only (PG, control), (2) commercially purchased unflavored solution with CBD, or (3) commercially purchased solutions with and without CBD and with different flavors. The in vitro toxicological effects were assessed using the following methods: (1) trypan blue exclusion assay (cell viability), (2) neutral red uptake assay (metabolic activity), and (3) ELISA (concentrations of inflammatory mediators). Results: Most flavored products with or without CBD were cytotoxic as compared to the air control. Overall, aerosols with CBD were more cytotoxic than aerosols without CBD irrelevant of the flavoring used in the product. Although, unflavored aerosols containing CBD in PG were significantly more cytotoxic than aerosols containing only PG, not all flavored products containing CBD were significantly more toxic than the same flavored products without CBD. Most CBD containing products significantly increase the concentration of cytokines released as compared to the same flavored products without CBD. Conclusion: Different flavors show different cytotoxic effects in CBD-containing e-cigarettes. Aerosols emitted from CBD containing e-cigarettes were more cytotoxic than those emitted from CBD-free e-cigarettes.

Effect of aerosolized nicotine on human bronchial epithelial cells is amplified after co-administration with cannabidiol (CBD): a pilot in vitro study.

BACKGROUND: Population-based studies suggest increasing rates of concurrent use of vaping products that contain either nicotine or cannabinoids. The aim of this pilot study was to test in vitro the acute inhalation toxicity of vaporized flavored and unflavored nicotine solutions co-administered with cannabidiol (CBD). METHODS: Bronchial epithelial cells (H292) were exposed directly to aerosol generated from electronic cigarettes refilled with propylene glycol only, unflavored nicotine solutions in propylene glycol with and without CBD, as well as to solutions containing only CBD. Cells were also exposed to a commercially available flavored solution containing nicotine and CBD. The in vitro toxicological effects were assessed after exposure using the following methods: 1) a trypan blue exclusion assay (cell viability), 2) neutral red uptake assay (metabolic activity) and 3) ELISA (concentrations of inflammatory mediators). RESULTS: Unflavored solution containing only CBD was significantly more cytotoxic than unflavored solution containing only nicotine. Unflavored solution containing both CBD and nicotine was significantly more cytotoxic than unflavored solutions with only nicotine. Levels of released cytokines were significantly higher when cells were co-exposed to nicotine and CBD as compared to cells exposed to only nicotine or only CBD. Overall, flavored products showed increased toxicity as compared to unflavored solutions. CONCLUSIONS: This pilot in vitro study suggests independent and additive toxic effects of vaporized nicotine and CBD. Observed toxic effects are accentuated by flavorings. Future studies are needed to determine the potential long-term health consequences of concurrent use of vaporized nicotine and cannabis products.

In Vitro Consequences of Electronic-Cigarette Flavoring Exposure on the Immature Lung.

Background: The developing lung is uniquely susceptible and may be at increased risk of injury with exposure to e-cigarette constituents. We hypothesize that cellular toxicity and airway and vascular responses with exposure to flavored refill solutions may be altered in the immature lung. Methods: Fetal, neonatal, and adult ovine pulmonary artery smooth muscle cells (PASMC) were exposed to popular flavored nicotine-free e-cigarette refill solutions (menthol, strawberry, tobacco, and vanilla) and unflavored solvents: propylene glycol (PG) or vegetable glycerin (VG). Viability was assessed by lactate dehydrogenase assay. Brochodilation and vasoreactivity were determined on isolated ovine bronchial rings (BR) and pulmonary arteries (PA). Results: Neither PG or VG impacted viability of immature or adult cells; however, exposure to menthol and strawberry flavored solutions increased cell death. Neonatal cells were uniquely susceptible to menthol flavoring-induced toxicity, and all four flavorings demonstrated lower lethal doses (LD50) in immature PASMC. Exposure to flavored solutions induced bronchodilation of neonatal BR, while only menthol induced airway relaxation in adults. In contrast, PG/VG and flavored solutions did not impact vasoreactivity with the exception of menthol-induced relaxation of adult PAs. Conclusion: The immature lung is uniquely susceptible to cellular toxicity and altered airway responses with exposure to common flavored e-cigarette solutions.

An exploration into "do-it-yourself" (DIY) e-liquid mixing: Users' motivations, practices and product laboratory analysis.

BACKGROUND: E-liquids are commercially available and manufactured, however some users of e-cigarettes prefer to prepare them at home (Do-it-Yourself; DIY) using individual ingredients. To date there is a paucity of research on how and why users make their own e-liquids. METHODS: Forty-one European and US based exclusive users of e-cigarettes (ex-smokers) were individually interviewed. Structured interviews focused on motivations for home-mixing, practices, buying habits and broader themes around reasons for long-term vaping. We also measured nicotine and solvent concentrations and analysed 33 DIY e-liquids collected from 16 participants for nicotine, solvents, flavourings, and potentially harmful chemicals. RESULTS: There were four main reasons for DIY: 1) economical (financial savings), 2) self-control over manufacturing process, 3) novelty, fun and 4) higher nicotine concentrations. Twelve out of 16 participants achieved nicotine concentration within 20% of their intended limit. Samples from five participants were above the EU Tobacco Products Directive's (TPD) 20 mg/ml nicotine concentration upper limit. Most samples contained more vegetable glycerine (VG) than propylene glycol (PG) and the most commonly used flavourings were dessert, e.g., vanilla and caramel. Chemical analysis also revealed presence of several potentially harmful chemicals and respiratory irritants, including cinnamaldehyde, benzaldehyde, and acetoin. CONCLUSION: DIY may offer users of e-cigarettes a long-term affordable practical method of vaping. Recommended safety advice needs to reflect actual and fast moving user behaviour.

Identification of flavouring chemicals and potential toxicants in e-cigarette products in Ontario, Canada.

OBJECTIVES: The current study examined constituents of e-cigarette products on the Canadian market, with a focus on the province of Ontario. METHODS: E-cigarettes were systematically purchased at 80 retail outlets across 4 cities in Ontario, Canada, in January-February 2015. Product constituents were identified using gas chromatography and mass spectrometry. Additionally, tobacco-specific nitrosamines (TSNAs) were quantified in tested products using liquid chromatography with tandem mass spectrometry. RESULTS: A total of 166 e-cigarette products were purchased, including disposable products (33%), refillable products (14%), and e-liquids (53%). Overall, e-cigarette products had an average of 6.2 (SD = 3.6) flavouring chemicals. E-cigarettes with sweet flavours (e.g., desserts, alcoholic drinks) had a significantly greater number of flavouring chemicals when compared with tobacco- and menthol-flavoured products (p < 0.05). Approximately one fifth (21%) of products contained flavouring chemicals with potential risk of inhalation toxicity (benzyl alcohol, benzaldehyde, vanillin). An additional 8 toxicants (e.g., acrolein, diacetyl) were detected in a total of 14 e-cigarette products. Measurable levels of TSNAs were detected in 70% of tested products. CONCLUSION: E-cigarettes purchased in Ontario, Canada, contained several constituents that may present excess risk, including some flavouring chemicals and carcinogenic nitrosamines. Further research is needed to determine whether the levels of these constituents have implications for the magnitude of risk to users. The findings reveal several policy gaps that may be addressed by developing regulatory product standards and labelling practices for e-cigarettes.

Cytotoxic effects of heated tobacco products (HTP) on human bronchial epithelial cells.

BACKGROUND: Heated tobacco product(s) (HTP), also called heat-not-burn products, are a re-emerging class of tobacco products that purport to reduce health risk compared with smoking combustible tobacco products. This study examined the potential toxic effects of inhaling emissions from an HTP in comparison with electronic and combustible tobacco cigarettes. METHODS: Inhalation toxicity of HTP (IQOS; tobacco flavour), e-cigarette (MarkTen; tobacco flavour) and tobacco cigarette (Marlboro Red) was examined in vitro using an air-liquid interface with human bronchial epithelial cells (H292). Cells were exposed directly to 55 puffs from the e-cigarette, 12 puffs from the HTP and 8 puffs from the tobacco cigarette to equilibrate nicotine delivery to the cells across products. Cytotoxicity was measured using neutral red uptake and trypan blue assays. Cytotoxic effects of each tested product (HTP, e-cigarette and tobacco cigarette) were compared with an air control. Release of inflammatory markers (cytokines) was measured using ELISA. RESULTS: The HTP showed higher cytotoxicity compared with the air controls using the neutral red assay. The HTP also showed higher cytotoxicity than the e-cigarette, but lower cytotoxicity than the combustible cigarettes using the same assay. A significant increase in cytokines levels, compared with air controls, was observed postexposure to tobacco smoke but not to emissions from HTP or e-cigarette aerosol. DISCUSSION: Using limited cytotoxic measures, the HTP showed reduced cytotoxicity relative to a combustible cigarette but higher toxicity than an e-cigarette. More comprehensive testing is needed to determine long-term effects of inhaling emissions from HTP.

Circadian Puffing Behavior and Topography Among E-cigarette Users.

OBJECTIVES: The aim of this study was to examine puffing behavior and topography over 24 hours among regular electronic cigarette (e-cigarette) users. METHODS: Twenty-four adult e-cigarette users (15 male) vaped their personal e-cigarettes ad-lib over the course of 24 hours. Participants took each puff via calibrated CReSS pocket topography monitors. We analyzed: number of puffs per day per session, mean puff volume, mean puff flow rate, mean duration of puff, and mean interval between puffs. RESULTS: Over 24 hours participants took on average 156.2±10.3 puffs, clustered in 10.2±7.9 puffs per puffing session with an average puff interval of 15.4±22.0 sec. A single puff lasted on average 3.0±1.2 sec, had a volume of 73.4±51.5 ml, and was taken with the average flow rate of 24.7±10.2 ml/sec. CONCLUSIONS: There is substantial variability among e-cigarette users in the way they puff on these devices. When e-cigarette aerosol is generated for laboratory studies, there is a need for validated puffing protocols that not only reflects the most-common pattern but also intensive puffing behaviors observed among some e-cigarette users.

Three-dimensional cultures of mouse submandibular and parotid glands: a comparative study.

Freshly isolated salivary cells can be plated on an extracellular matrix, such as growth factor-reduced Matrigel (GFR-MG), to induce the formation of three-dimensional (3D) structures. Cells grown on GFR-MG are able to form round structures with hollow lumina, capable of sustaining amylase expression. In contrast, cells grown on plastic do not exhibit these features. Our recent studies have used mouse parotid gland (PG) cells, grown on different extracellular matrices, as a model for acinar formation. However, PG cells were not able to respond to the secretory agonist carbachol beyond 5 days and did not sustain polarity over time, regardless of the substratum. An alternative option relies in the use of mouse submandibular glands (SMG), which are more anatomically accessible and yield a larger number of cells. We compared SMG and PG cell clusters (partially dissociated glands) for their ability to form hollow round structures, sustain amylase and maintain secretory function when grown on GFR-MG. The results were as follows: (a) SMG cell clusters formed more organized and larger structures than PG cell clusters; (b) both SMG and PG cell clusters maintained α-amylase expression over time; (c) SMG cell clusters maintained agonist-induced secretory responses over time; and (d) SMG cell clusters maintained secretory granules and cell-cell junctions. These results indicate that mouse SMG cell clusters are more amenable for the development of a bioengineered salivary gland than PG cell clusters, as they form more organized and functional structures. Copyright © 2014 John Wiley & Sons, Ltd.