Levels Of Tobacco-specific Nitrosamines Research Articles

Impact of More Intense Smoking Parameters and Flavor Variety on Toxicant Levels in Emissions of a Heated Tobacco Product.

IQOS HEETS are promoted as reduced-risk alternatives to cigarettes. Although some studies have investigated the chemical composition of HEETS emissions, little is known on whether toxicant levels in such emissions are affected by different puffing parameters and flavor varieties. This has important implications when assessing actual human exposure, since IQOS users develop a specific and personalized puffing behavior and may use different HEETS variants. This study measured the levels of nicotine, total particulate matter, carbonyl compounds, and tobacco-specific nitrosamines (TSNAs) in the emissions of nine differently flavored HEETS and two cigarettes (1R6F and Marlboro Red, MR). Emissions from Yellow HEETS, 1R6F, and MR were collected using the World Health Organization Intense smoking regime and four more intense smoking regimes. Yellow HEETS aerosol contained lower levels of toxicants compared to 1R6F and MR smoke. More intense smoking regimes increased carbonyl release in cigarette smoke, whereas only higher puff frequency led to lower levels of toxicants in Yellow HEETS aerosol. Some HEETS varieties exhibited higher levels of formaldehyde and TSNAs in their aerosols compared to Yellow HEETS. Puff frequency was identified as the only smoking parameter that significantly lowered the release of almost all toxicants in Yellow HEETS, whereas a combination of higher puff volume and puff duration led to increased levels of some carbonyls. Differences in toxicant levels between various commercially available HEETS have important implications when assessing their health impact, as their consumption might induce different toxicant exposure and health effects. HEETS release about half as much nicotine and substantially lower levels of toxicants compared to cigarettes. Literature data showed that puffing intensity is increased in cigarette smokers switching to HEETS, maybe in reaction to these lower nicotine levels. Our results show a differential impact of increased puff frequency, puff duration, and puff volume in the release of toxicants from HEETS. Thus, industry-independent studies on puff topography are critical to make choices for the most relevant puffing regime for heated tobacco product regulation. Regulators should consider evaluating the health impact of multiple HEETS varieties, as the tobacco filler composition significantly affects the release of certain toxicants.

Reducing nitrate and tobacco-specific nitrosamine level in burley tobacco leaves through grafting on flue-cured tobacco rootstock.

Nitrosation of pyridine alkaloids in tobacco generates tobacco-specific nitrosamines (TSNAs), which are notable toxicants in tobacco products and smoke. Burley tobacco, a chloroplast- and nitrogen (N)-deficient phenotype that accumulates high levels of nitrate-nitrogen (NO3-N) in its leaves, is particularly susceptible to TSNAs formation. In this study, reciprocal pot and field grafting experiments were conducted using burley tobacco Eyan No.1 and flue-cured tobacco K326 to investigate whether grafting burley tobacco scions on flue-cured tobacco rootstocks could enhance pigment biosynthesis and photosynthesis, while reducing the NO3-N level in burley tobacco leaves. Grafting burley tobacco scions on flue-cured tobacco rootstocks significantly increased the total pigment content, photosynthetic rate, biomass, nitrate reductase and glutamine synthetase activities, as well as ammonium-nitrogen (NH4-N), total soluble and reducing sugar, and soluble protein levels in burley tobacco leaves compared with burley tobacco self-rooting, while decreasing the NO3-N level and nitrate-N to total N ratio. Transcriptomic analysis revealed that grafting resulted in upregulated expression of genes involved in starch, sucrose, porphyrin, chlorophyll, and N metabolism, as well as carbon fixation and carotenoid biosynthesis. The findings suggest that grafting on high N use efficiency rootstock is an exceptionally promising means of decreasing NO3-N accumulation by improving photosynthesis and N metabolism in the scion, thereby reducing the levels of harmful TSNAs.

Exposure to Tobacco-Specific Nitrosamines Among People Who Vape, Smoke, or do Neither: A Systematic Review and Meta-Analysis.

Smoking exposes people to high levels of Tobacco-Specific Nitrosamines (TSNAs), which include potent carcinogens. We systematically reviewed TSNA exposure between people smoking, vaping, and doing neither. Databases were searched between August 2017-March 2022, using vaping-related terms. Peer-reviewed articles reporting TSNA metabolites (NNAL, NNN, NAB, and NAT) levels in bio-samples among adults exclusively vaping, exclusively smoking, or doing neither were included. Where possible, meta-analyses were conducted. Of 12 781 identified studies, 22 were included. TSNA levels fell substantially when people who smoke switched to vaping in longitudinal studies and were lower among people who vaped compared to smoked in cross-sectional studies. Levels of TSNAs were similar when comparing people who switched from smoking to vaping, to those who switched to no use of nicotine products, in longitudinal studies. Levels were higher among people who vaped compared to people who neither vaped nor smoked in cross-sectional studies.When comparing people who vaped to smoked: pooled urinary NNAL was 79% lower across three randomized controlled trials and 96% lower across three cross-sectional studies; pooled NAB was 87% lower and NAT 94% lower in two cross-sectional studies. When comparing people who neither vaped nor smoked to people who vaped, pooled urinary NNAL was 80%, NAB 26%, and NAT 27% lower in two cross-sectional studies. Other longitudinal data, and NNN levels could not be pooled. Exposure to all TSNAs was lower among people who vaped compared to people who smoked. Levels were higher among people who vaped compared to people who neither vaped nor smoked. As well as TSNAs, there are many other toxicant exposures from smoking and vaping that can increase the risk of disease. However, it is likely that the reduced exposure to TSNAs from vaping relative to smoking reduces the risk to health of those who use vaping products to quit smoking. Future high-quality research, with robust definitions of exclusive vaping and smoking, and accounting for TSNAs half-lives, is needed to fully assess exposure to TSNAs among people who vape.

Tobacco Nitrate and Free Radical Levels in the Mainstream Smoke of US Cigarette Brands.

Tobacco nitrate levels have been known to impact the levels of toxicants such as polyaromatic hydrocarbons and tobacco-specific nitrosamines (TSNAs) produced during smoking. Recent work in our group showed that the intrinsic nitrate levels in individual tobacco varieties also have a large influence on the formation of gas-phase (GP) free radicals in the mainstream smoke of cigarettes produced with a single tobacco variety. As tobacco nitrate content is a potential target for future regulatory policies, we investigated whether the levels of GP free radicals in the smoke from commercially available cigarettes is also dependent on the nitrate content in the corresponding tobacco blends. GP and particulate-phase (PP) free radical yields in mainstream smoke produced from 25 popular US cigarette brands were measured by electron paramagnetic resonance (EPR) spectroscopy. For each brand, we also measured levels of the TSNAs NNN (N'-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) via HPLC-MS and the nicotine content via GC-FID. Our results show that the intrinsic nitrate levels varied >15-fold and GP radicals varied 4-fold among the 25 brands tested. The GP radicals were correlated with intrinsic nitrate levels (r = 0.87, p < 0.001). NNK and NNN levels varied >8-fold and 12-fold, respectively. We found that NNK was moderately correlated to nitrate content (r = 0.42, p = 0.03) while the NNN was strongly correlated to the nitrate content (r = 0.65, p < 0.001). Nicotine levels varied the least (<3-fold) but showed a moderate negative correlation to nitrate content (r = -0.47, p = 0.02). No statistically significant correlation was observed between nicotine and TSNA levels in mainstream smoke. Overall, this demonstrates that the nitrate content of tobacco blends used in US cigarette brands impacts toxicant output in the mainstream smoke, although other proprietary variables (total ventilation, additives, filter type, etc.) may also modulate these results.

UHPLC-MS/MS method for the simultaneous determination of nicotine and tobacco-specific nitrosamines NNN and NNK for use in preclinical studies

A new method was developed and validated for the simultaneous determination of nicotine and tobacco-specific nitrosamines (TSNAs) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in two different tests matrices: porcine buccal epithelium tissue and phosphate buffered saline (PBS) extracts of smokeless tobacco products. The novelty of this work is in the development of a liquid chromatography tandem mass spectrometry method that can provide simultaneous quantification of trace levels of TSNAs and high concentrations of nicotine in biological media. Precision, accuracy, and stability were evaluated during method validation to ensure the method was fit for purpose. Several sample preparation and extraction methods were evaluated to minimize matrix effects and maximize analyte recoveries. The method was accurate in the range of 81.1% – 117%; repeatability was estimated in the range of 1.5% – 13.6% across multiple concentrations. The linear regression correlation coefficient (R2) was greater than 0.9959 for all analytes, and the limit of detection (LOD) was determined for nicotine, NNK, and NNN at 1 ng/mL 0.005 ng/mL, and 0.006 ng/ mL, respectively. Our method was found to be appropriate for the analysis of nicotine, NNN, and NNK in the porcine buccal epithelium and PBS extracts of smokeless tobacco products.Graphical abstract

A chemometric experimental design with three-step stacking capillary electrophoresis for analysis of five tobacco-specific nitrosamines in cigarette products

Tobacco-specific nitrosamines (TSNAs) as carcinogens endanger our health and life from cigarette products. However, the safe range of TSNAs levels in commercial cigarette products has not yet been established. For the purpose of safety and supervision, a three-step stacking approach including field amplified sample injection (FASI), sweeping, and analyte focusing by micelle collapse (AFMC), was developed for the simultaneous determination of five TSNAs levels in cigarette products. This approach also involved aspects of chemometric experimental design, including fractional factorial design and central composite design. After the multilevel optimization of the experimental design, the five TSNAs were well separated. The LOD (S/N = 3) values of the N´-nitrosonornicotine (NNN), N´-nitrosoanatabine (NAT), N´-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the FASI-sweeping-AFMC CE approach were 1.000 ng/mL, 0.500 ng/mL, 0.125 ng/mL, 1.000 ng/mL, and 0.500 ng/mL respectively. The results of relative standard deviation (RSD) and relative error (RE) were all less than 3.35%, demonstrating good precision and accuracy. Finally, this novel approach was further applied to monitor three commercial cigarette products, and a range of 250.1–336.6 ng/g for NNN, 481.6–526.7 ng/g for NAT, 82.2–247.6 ng/g for NAB, 167.7–473.7 ng/g for NNAL, and 39.4–246.7 ng/g for NNK could be observed among these. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of five TSNAs levels in cigarette products and could serve as a tool for assays of quality control of nitrosamines.

Influence of Nitrite on Formation of Tobacco-Specific Nitrosamines in Electronic Cigarette Liquids and Aerosols.

Tobacco-specific nitrosamine (TSNA) formation occurred during aerosol generation from select commercial cig-a-like e-cigarette products. To understand the drivers behind the potential formation of TSNAs in electronic cigarette (e-cigarette) aerosols and e-liquids, model e-liquid systems were generated in the lab to demonstrate that nitrite can react with nicotine and minor alkaloids to form TSNAs in e-liquids. In the presence of nitrite and nicotine, TSNA levels in e-liquids increased over time and the process was accelerated by elevated temperature. Additionally, TSNAs formed during aerosol generation when nitrite was present in the corresponding e-liquids. The commercial e-cigarette products that showed higher levels and formation of TSNAs were observed to contain nitrite and minor alkaloid impurities in the corresponding e-liquids. This study provides valuable information about drivers for TSNA formation in e-liquids and e-cigarette aerosols that may be applied to the evaluation and quality assurance of e-cigarette products.

CLCNt2 Mediates Nitrate Content in Tobacco Leaf, Impacting the Production of Tobacco-Specific Nitrosamines in Cured Leaves.

Nitrate accumulation in tobacco (Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies.

Online In-Tube Solid-Phase Microextraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Tobacco-Specific Nitrosamines in Hair Samples.

Active and passive smoking are serious public health concerns Assessment of tobacco smoke exposure using effective biomarkers is needed. In this study, we developed a simultaneous determination method of five tobacco-specific nitrosamines (TSNAs) in hair by online in-tube solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC–MS/MS). TSNAs were extracted and concentrated on Supel-Q PLOT capillary by in-tube SPME and separated and detected within 5 min by LC–MS/MS using Capcell Pak C18 MGIII column and positive ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. The calibration curves of TSNAs showed good linearity in the range of 0.5–1000 pg mL–1 using their stable isotope-labeled internal standards. Moreover, the limits of detection (S/N = 3) of TSNAs were in the range of 0.02–1.14 pg mL–1, and intra-day and inter-day precisions were below 7.3% and 9.2% (n = 5), respectively. The developed method is highly sensitive and specific and can easily measure TSNA levels using 5 mg hair samples. This method was used to assess long-term exposure levels to tobacco smoke in smokers and non-smokers.

The ultra-structural, metabolomic and metagenomic characterisation of the sudanese smokeless tobacco 'Toombak'.

Toombak is a smokeless tobacco produced from the Nicotiana rustica tobacco plant from Sudan. Pre-prepared and ready to buy Toombak samples were analysed using mass spectrometry (heavy metals), gas and liquid chromatography (metabolomics), 16S rRNA metagenomic sequencing (microbiome) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX) and pH analysis. Chromium, cobalt, and copper were high in the pre-prepared form of Toombak while iron, tobacco specific nitrosamines (TSNAs), formaldehyde and acetaldehyde were high in both types. Firmicutes and Actinobacteria dominated Toombak. Samples of ready to buy Toombak showed inter-variational differences depending on place of purchase. We found Virgibacillus were increased in the pre-prepared form while Corynebacterium casei, Atopococus tabaci, Atopostipes suicloacalis, Oceanobacillus chironomi and Staphylococcus gallinarum were the most abundant species in the ready to buy forms. PICRUSt analysis highlighted increased activity of metal transport systems in the ready to buy samples as well as an antibiotic transport system. SEM-EDX highlighted large non-homogenous, irregular particles with increased sodium, while pH of samples was in the alkaline range. The final composition of Toombak is affected by its method of preparation and the end product has the potential to impart many negative consequences on the health of its users. TSNA levels observed in Toombak were some of the highest in the world while the micro-environment of Toombak supports a distinct microbiota profile.

Tobacco-Specific Nitrosamines (NNAL, NNN, NAT, and NAB) Exposures in the US Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).

The tobacco-specific nitrosamines (TSNAs) are an important group of carcinogens found in tobacco and tobacco smoke. To describe and characterize the levels of TSNAs in the Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014), we present four biomarkers of TSNA exposure: N'-nitrosonornicotine, N'-nitrosoanabasine, N'-nitrosoanatabine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which is the primary urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. We measured total TSNAs in 11 522 adults who provided urine using automated solid-phase extraction coupled to isotope dilution liquid chromatography-tandem mass spectrometry. After exclusions in this current analysis, we selected 11 004 NNAL results, 10 753 N'-nitrosonornicotine results, 10 919 N'-nitrosoanatabine results, and 10 996 N'-nitrosoanabasine results for data analysis. Geometric means and correlations were calculated using SAS and SUDAAN. TSNA concentrations were associated with choice of tobacco product and frequency of use. Among established, every day, exclusive tobacco product users, the geometric mean urinary NNAL concentration was highest for smokeless tobacco users (993.3; 95% confidence interval [CI: 839.2, 1147.3] ng/g creatinine), followed by all types of combustible tobacco product users (285.4; 95% CI: [267.9, 303.0] ng/g creatinine), poly tobacco users (278.6; 95% CI: [254.9, 302.2] ng/g creatinine), and e-cigarette product users (6.3; 95% CI: [4.7, 7.9] ng/g creatinine). TSNA concentrations were higher in every day users than in intermittent users for all the tobacco product groups. Among single product users, exposure to TSNAs differed by sex, age, race/ethnicity, and education. Urinary TSNAs and nicotine metabolite biomarkers were also highly correlated. We have provided PATH Study estimates of TSNA exposure among US adult users of a variety of tobacco products. These data can inform future tobacco product and human exposure evaluations and related regulatory activities.

Comparison of tobacco-specific nitrosamine levelsin smokeless tobacco products: High levels in products from Bangladesh.

Bangladesh exhibits the second highest rate of smokeless tobacco (SLT) product usage in the world, and this has been associated with the high upper aerodigestive tract cancer incidence in this country. The goal of the present study was to examine the levels of the highly carcinogenic tobacco-specific nitrosamines (TSNAs) in Bangladeshi SLT products and compare these levels to that observed in SLT brands from southeast Asia and the USA. The levels of TSNAs and nicotine were determined by LC-MS/MS in twenty-eight SLT brands and several tobacco additives from Bangladesh, as well as several SLT brands from India, Pakistan and the USA. The levels of N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB) in Bangladeshi SLT brands ranged from 1.1–59, 0.15–34, 0.79–45, and 0.037–13 μg/g SLT powder, respectively. The mean levels of the highly carcinogenic TSNAs (NNN+NNK) were 7.4-, 2.4-, and 63-fold higher in Bangladeshi SLT products as compared to SLT brands from the USA, India and Pakistan, respectively; these trends were also observed for NAT and NAB. Similar mean levels of nicotine were observed in the Bangladeshi brands (31 mg/g powder) versus brands from the USA (25 mg/g powder) and India (20 mg/g powder); they were 3-fold higher than brands from Pakistan (10 mg/g powder). Gul SLT brands exhibited the highest pH and the highest levels of unprotonated nicotine. The high levels of TSNAs in Bangladeshi SLT brands may be an important factor contributing to the high rates of upper aerodigestive tract cancer in Bangladesh.

Effect of Ordering Method on Tobacco-Specific Nitrosamines (TSNAs) Content in Dark Air-Cured and Burley Tobacco

Tobacco-specific nitrosamines (TSNAs) are known carcinogens in cured tobacco. They are produced primarily during the curing process, but agronomic practices occurring in the field as well as handling practices after curing may also influence TSNA levels, particularly if cured leaf is stored at high moisture. After curing and during market preparation, the cured leaf must be supple to avoid breakage. Ideally, this is after a period of wet weather during which the leaf absorbs moisture and comes into order or case. Often the weather remains dry for long periods after curing, and growers resort to artificial ordering to take down a sufficient amount of their crop to work on for several days, during which time the tobacco is bulked. The effect of this artificial ordering on TSNAs during short-term storage is not known. Field experiments were conducted in each of 3 years at two locations in Kentucky to evaluate TSNA accumulation following several ordering methods in dark air-cured and burley tobacco types. Treatments included natural ordering and variants of steaming and misting, which are both commonly used artificial ordering methods. At the Princeton location, samples were taken within 24 hr after the ordering treatments were done. In Lexington, samples were taken sequentially at takedown, after ordering, and after 14 d in the bulk. There were limited and inconsistent differences in total TSNAs between methods of ordering, and the TSNA levels were not affected by the moisture content of the leaf during bulking. There was a significant increase in TSNAs in the 24-hr period between takedown and bulking, which cannot be explained. We conclude that, in Kentucky, growers should use ordering methods that are best suited for their production system, but this may not be the case in warmer climates.

Variability of TSNA in U.S. Tobacco and Moist Smokeless Tobacco Products

Tobacco-specific nitrosamines (TSNAs) have been of concern to the public health community for decades and their reduction through agricultural practices, plant breeding, and tobacco processing has also been a decades-long industry effort. Despite those efforts, TSNAs, though lower, continue to be constituents of concern in tobacco products. This paper examines the TSNA levels of dark air-cured, dark fire-cured, and burley tobaccos purchased in the United States by U.S. Smokeless Tobacco Company LLC (USSTC) and of nine finished USSTC moist smokeless tobacco products. TSNA values of the incoming purchased tobaccos and the finished products showed considerable variability. For the incoming tobaccos, the coefficient of variation was generally more than 100 % for each tobacco type and for each of the measured TSNAs. The relative TSNA variability of the finished tobacco products was also considerable, averaging approximately 25 %. It was also found that the measured values for the finished products averaged well above the proposed FDA NNN proposed product standard of 1.0 μg/g dry weight. Because of the large variability in NNN values, products would have to average well below FDA’s proposed product standard to be consistently compliant.

Use of electronic nicotine delivery systems by pregnant women II: Hair biomarkers for exposures to nicotine and tobacco-specific nitrosamines.

INTRODUCTIONPublic awareness of electronic nicotine delivery systems (ENDS) has increased over time, and the perception that ENDS offer a safer alternative to cigarettes may lead some pregnant women to use them to reduce cigarette smoking during pregnancy. No previous studies have used metabolite levels in hair to measure nicotine exposure for ENDS users during pregnancy. We aimed to measure and compare levels of nicotine, cotinine, and tobacco-specific nitrosamines (TSNAs) in hair samples from pregnant women who were current ENDS users, current smokers, and current non-smokers. We also aimed to estimate the association between ENDS use/smoking and smallness for gestational age (SGA).METHODSWe used hair specimens from pregnant women who were dual users (ENDS and cigarettes), smokers, and non-smokers from a prospective cohort study to estimate exposure to nicotine, cotinine, and TSNAs. The exposure biomarkers and self-reports of smoking and ENDS use were used in log-binomial regression models to estimate risk ratios (RRs) for SGA among offspring.RESULTSNicotine concentrations for pregnant dual users were not significantly different from those for smokers (11.0 and 10.6 ng/mg hair, respectively; p=0.58). Similarly, levels of cotinine, and TSNAs for pregnant dual users were not lower than those for smokers. The RR for SGA was similar for dual users and smokers relative to nonsmokers, (RR=3.5, 95% CI: 0.8–14.8) and (RR=3.3, 95% CI: 0.9–11.6), respectively. Using self-reports confirmed by hair nicotine, the RR values for dual ENDS users and smokers were 8.3 (95% CI: 1.0–69.1) and 7.3 (95% CI:1.0–59.0), respectively.CONCLUSIONSWe did not observe lower levels of nicotine, cotinine, and TSNAs for current dual users compared to smokers during pregnancy. The risk of SGA for offspring of pregnant dual users was similar to that for offspring of pregnant smokers. Future studies are needed to further estimate the magnitude of the association between ENDS use and smallness for gestational age.

The Health Risks of Electronic Cigarette Use to Bystanders.

This works aimed to assess the health risks of e-cigarette use to bystanders. The exhaled breath of 17 volunteers was collected while they were vaping, and the levels of nicotine, propylene glycol, glycerol, formaldehyde, acetaldehyde, acrolein, tobacco-specific nitrosamines (TSNAs), and heavy metals were analyzed. Increased levels of nicotine, propylene glycol, TSNAs and copper were found in the exhaled breath of the volunteers. From these measurements, bystander exposure was estimated for two different scenarios: (1) A non-ventilated car with two e-cigarette users and (2) a ventilated office with one e-cigarette user. Our results show that bystanders may experience irritation of the respiratory tract as a result of exposure to propylene glycol and glycerol. Systemic effects of nicotine should also be expected if nicotine-containing e-liquid is used, including palpitations, and an increase of the systolic blood pressure. Furthermore, due to the presence of TSNAs in some e-liquids, an increased risk of tumors could not be excluded for the ‘car’ scenario. While e-cigarette use can clearly have effects on the health of bystanders, the risks depend on the rate of ventilation, dimensions of the room, and vaping behavior of the e-cigarette user. The presence of TSNAs in e-liquids can be avoided, which will prevent the most serious effect identified (increased risk of tumors).

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

The current study examined constituents of e-cigarette products on the Canadian market, with a focus on the province of Ontario. 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. 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 onefifth (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. 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.

Evaluation of tobacco‐specific nitrosamine (TSNA) content in smokeless tobacco products in Bangladesh

Head and neck cancers are among the most common cancers in Southeast Asia and are likely to face sharp increases in the coming years due to the widespread use of smokeless tobacco (SLT) products. Bangladesh has the second highest oral cancer mortality rate as well as the second highest rate of SLT product usage worldwide. SLT products contain high levels of the highly carcinogenic tobacco‐specific nitrosamines (TSNAs); they include N‐nitrosonornicotine (NNN), 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), N‐nitrosoanatabine (NAT), and N‐nitrosoanabasine (NAB). To date, the amount of TSNAs present in Bangladeshi SLT products has not been reported. The goal of the present study was to develop an efficient TSNA and nicotine extraction method to quantify the levels of TSNAs and nicotine present in 31 Bangladeshi SLT products. The concentration of NNN, NNK, NAT, and NAB in Bangladeshi SLT brands ranged from 1.07–59.18 μg/g SLT, 0.15–33.70 μg/g SLT, 0.79–44.64 μg/g SLT, and 0.03–12.85 μg/g SLT, respectively. Pan Masala, which is labeled as a non‐tobacco product, also contains low levels of TSNAs. Betel nut, a common flavor additive in SLT products in Bangladesh, contained no detectible TSNA levels. Bangladeshi SLT products contained significantly higher (p&lt;0.05) levels of TSNAs as compared to SLT brands from the United States (U.S.), and were higher on average than the levels observed in brands in other regions of southern Asia. NNN and NNK levels were 16 and 19, 2 and 3.5, and 62 and 85 fold higher than U.S., Indian and Pakistani SLT brands, respectively. Additionally, the levels of NNN and NNK measured in U.S. brands in this study were 1.2‐fold higher than previously reported values from 2013. This study demonstrates that the levels of TSNAs, including the major tobacco carcinogens NNN and NNK, are far higher in Bangladeshi SLT brands than they are in other regions of the world. Exposure to such high levels of carcinogens may be the most important contributing factor to the rate of head and neck as well as other tobacco‐related cancers observed in Bangladesh.Support or Funding InformationThis work is supported in part by grants from NIH, National Cancer Institute (grant R01‐ES025460, to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292, to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biochemical Profiling of Smokeless Tobacco Product Kiwam at Different Processing Steps

Introduction: Kiwam (qiwam) is a partially fermented tobacco product consumed with betel quid (paan). The major constituents of this product are tobacco, saffron (zaffrani) and some other additives. It contains tobacco-specific nitrosamines (TSNA) which is considered as a cancer causing agent. To elucidate the carcinogenic property of kiwam, biochemical profiling of its constituents at different stages of processing is needed. The major processing steps involved in the formation of kiwam and biochemical profiling/changes at each processing step is still unknown. Aim: To describe the major processing steps and biochemical changes that occur at each processing step during the preparation of kiwam. Methods: Tobacco leaves and stems were washed with Millipore water so as to remove the dirt particles from the leaves and stems. It is then boiled in water followed by filtering of the constituents to remove the leaves and stem residues. The filtrate was again boiled to form a thick paste residue. The resultant paste was partially fermented through sun curing, and lastly, saffron along with specific additives was added. The samples from each step were analyzed for biochemical profiling through Continuous Flow Autoanalyzer using Flow View Solution 3700 Analyzer (version 1.2.2) software. Results: The biochemical changes at TSNA levels were observed at each processing steps. The detailed chemical profiling will be presented during the meeting. Conclusion: Kiwam is rich in TSNA and hence its use should be avoided.

Tobacco-specific nitrosamines: A literature review

Cigarette smoke is a complex mixture of chemicals, including several tobacco-specific nitrosamines (TSNA). Most TSNA are formed in tobacco during the post-harvest period, while a number are produced when a cigarette is burned. Considerable evidence supports the role of TSNA important causative factors for cancers of the lung, pancreas, esophagus, and oral cavity in people who use tobacco products. Of the known TSNA, nicotine-derived nitrosamine ketone (NNK) and N-nitrosonornicotine (NNN) are the most carcinogenic. Other TSNA include N′-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB). New tobacco products (e.g., e-cigarettes) designed to attract consumers who are concerned about the health effects of tobacco have been appearing on the market. Several studies have reported that certain TSNA have been detected in the replacement liquids and vapour of e-cigarettes, but the levels are generally considerably lower than in tobacco cigarettes. Additionally, the FDA recently announced its intention to regulate TSNA in e-cigarettes, cigar tobacco and pipe tobacco. With the rise of new technologies for reducing the use of tobacco products—such as e-cigarettes— to evaluate exposure levels to these harmful chemicals over time, researchers will be monitoring levels of TSNA in the body as a result of the use of these devices.