Assessment of toxicological validity using tobacco emission condensates: A comparative analysis of emissions and condensates from 3R4F reference cigarettes and heated tobacco products.

The tobacco emission condensate, henceforth referred to as "tobacco condensate," plays a critical role in assessing the toxicity of tobacco products. This condensate, derived from tobacco emissions, provides an optimized liquid concentrate for storage and concentration control. Thus, the validation of its constituents is vital for toxicity assessments. This study used tobacco condensates from 3R4F cigarettes and three heated tobacco product (HTP) variants to quantify and contrast organic compounds (OCs) therein. The hazard index (HI) for tobacco emissions and condensates was determined to ascertain the assessment validity. The total particulate matter (TPM) for 3R4F registered at 17,667 µg cig-1, with its total OC (TOC) at 3777 µg cig-1. HTPs' TPM and TOC were 9342 ± 1918 µg cig-1 and 5258 ± 593 µg stick-1, respectively. 3R4F's heightened TPM likely arises from tar, while HTPs' OC concentrations are influenced by vegetable glycerin (2236-2688 µg stick-1) and propylene glycol (589-610 µg stick-1). During the condensation process, a substantial proportion of OCs in 3R4F smoke underwent significant concentration decreases, in contrast to HTPs, where fewer than half of the examined OCs exhibited notable concentration declines. The HI for tobacco emissions exhibited a marginally higher value compared to tobacco condensate, with variations ranging from 7.92% (HTPs) to 18.6% (3R4F), denoting a minimal differential. These observations emphasize the importance of accurate OC recovery techniques to maintain the validity and reliability of toxicity assessments based on tobacco condensates. This study not only deepens the comprehension of chemical behaviors in tobacco products but also establishes a novel benchmark for their toxicity evaluation, with profound implications for public health strategies and consumer protection.

210Po and 210Pb radioactivity levels in local and imported tobacco used in Palestine and Jordan.

This study aimed to assess the concentrations of 210Po and 210Pb in various tobacco samples from Palestine and Jordan. Cigarette smoking is recognized as a significant contributor to the radiation dose received by individuals, primarily due to the elevated levels of 210Pb and 210Po found in tobacco. The analysis revealed that the average concentrations of 210Po in locally sourced tobacco and cigarette samples in Palestine are 16.8 ± 2.3 mBq/g and 18.5 ± 2.0 mBq/g, with a total average of 17.8 ± 7.4 mBq/g (15.5 mBq/cigarette). Similarly, the average concentrations of 210Pb in these samples are 18.5 ± 2.6 mBq/g and 20.3 ± 2.2 mBq/g, with a total average of 19.6 ± 8.1 mBq/g (17.0 mBq/cigarette). In Jordan, the average concentrations of 210Po in cigarette samples and narghile tobacco are 20.1 ± 2.4 mBq/g and 18.3 ± 4.1 mBq/g, with a total average value of 19.6 ± 9.9 mBq/g (18.0 mBq/cigarette), while the average concentrations of 210Pb are 22.2 ± 2.6 mBq/g and 20.2 ± 4.5 mBq/g, with a total average value of 21.6 ± 10.8 mBq/g (19.9 mBq/cigarette). The annual effective doses resulting from inhalation were calculated for smokers of these samples. The findings revealed that the levels of 210Po and 210Pb radioactivity in certain investigated samples exceeded the results of studies in many countries of the world. The associated effective doses per year from smoking for all brands products in Palestine range from 34.7 µSv/y to 186.5 µSv/y with an average of 109.5 µSv/y, while in Jordan 54.5 µSv/y to 289.1 µSv/y with an average of 130.9 µSv/y.

Underreporting of non-study cigarette use by study participants confounds the interpretation of results from ambulatory clinical trial of reduced nicotine cigarettes.

BACKGROUND: As part of its comprehensive plan to significantly reduce the harm from tobacco products, the US Food and Drug Administration is establishing a product standard to lower nicotine in conventional cigarettes to make them "minimally addictive or non-addictive". Many clinical studies have investigated the potential impact of such a standard on smoking behavior and exposure to cigarette constituents. These ambulatory studies required participants who smoke to switch to reduced nicotine study cigarettes. In contrast to clinical trials on pharmaceuticals or medical devices, participants had ready access to non-study conventional nicotine cigarettes and high rates of non-study cigarette use were consistently reported. The magnitude of non-compliance, which could impact the interpretation of the study results, was not adequately assessed in these trials. METHODS: We conducted a secondary analysis of data from a large, randomized trial of reduced nicotine cigarettes with 840 participants to estimate the magnitude of non-compliance, i.e., the average number of non-study cigarettes smoked per day by study participants assigned to reduced nicotine cigarettes. Individual participants' non-study cigarette use was estimated based on his/her urinary total nicotine equivalent level, the nicotine content of the study cigarette assigned and the self-reported number of cigarettes smoked, using a previously published method. RESULTS: Our analysis showed that (1) there is a large variation in the number of non-study cigarettes smoked by participants within each group (coefficient of variation 90-232%); (2) participants in reduced nicotine cigarette groups underreported their mean number of non-study cigarettes smoked per day by 85-91%; and (3) the biochemical-based estimates indicate no reduction in the mean number of total cigarettes smoked per day for any group assigned to reduced nicotine cigarettes after accounting for non-study cigarettes. CONCLUSIONS: High levels of non-compliance, in both the rate and magnitude of non-study cigarette use, are common in ambulatory reduced nicotine cigarette trials where participants have access to conventional nicotine non-study cigarettes. The potential impact of high non-compliance on study outcomes should be considered when interpreting the results from such ambulatory studies.

Synthetic Cooling Agents in Australian-Marketed E-cigarette Refill Liquids and Disposable E-cigarettes: Trends Follow the U.S. Market.

INTRODUCTION: E-cigarettes are becoming increasingly popular in Australia, especially amongst the younger population. The synthetic cooling molecules WS-3 and WS-23 have been identified in e-cigarette products from the United States and Europe. The extent of inclusion of these synthetic coolants in Australian e-liquids is unknown, particularly in newer disposable e-cigarettes. AIMS AND METHODS: E-cigarettes and e-liquids were purchased within Australia and anonymously donated by Australian users. Nicotine, WS-3, WS-23, and menthol were quantified in the e-liquids using gas chromatography-mass spectrometry (GC-MS). RESULTS: WS-23 and nicotine were detected in all of the disposable e-cigarettes with WS-23 often present in high concentrations. There was no correlation between cooling terms in the flavor name and the inclusion of cooling agents. Only three bottled e-liquids were found to contain WS-23 while none contained WS-3 above the limit of detection. CONCLUSIONS: Synthetic coolants were a common addition in disposable e-cigarettes while rarely added to e-liquid bottle refills. Their inclusion in these products is reflective of trends observed in United States and European e-cigarette products. IMPLICATIONS: The increase in synthetic cooling agents as components of e-liquids, particularly disposable e-cigarette devices, has been observed within Australian samples across a range of brands and flavors. WS-23 was present in every disposable e-cigarette analyzed in this study, often in relatively high concentrations. Its inhalational toxicology should be considered when evaluating the safety of these products.

Heterogeneous deposition of regular and mentholated little cigar smoke in the lungs of Sprague-Dawley rats.

BACKGROUND: Quantifying the dose and distribution of tobacco smoke in the respiratory system is critical for understanding its toxicity, addiction potential, and health impacts. Epidemiologic studies indicate that the incidence of lung tumors varies across different lung regions, suggesting there may be a heterogeneous deposition of smoke particles leading to greater health risks in specific regions. Despite this, few studies have examined the lobar spatial distribution of inhaled particles from tobacco smoke. This gap in knowledge, coupled with the growing popularity of little cigars among youth, underscores the need for additional research with little cigars. RESULTS: In our study, we analyzed the lobar deposition in rat lungs of smoke particles from combusted regular and mentholated Swisher Sweets little cigars. Twelve-week-old male and female Sprague-Dawley rats were exposed to smoke particles at a concentration of 84 ± 5 mg/m3 for 2 h, after which individual lung lobes were examined. We utilized Inductively Coupled Plasma Mass Spectrometry to quantify lobar chromium concentrations, serving as a smoke particle tracer. Our findings demonstrated an overall higher particle deposition from regular little cigars than from the mentholated ones. Higher particle deposition fraction was observed in the left and caudal lobes than other lobes. We also observed sex-based differences in the normalized deposition fractions among lobes. Animal study results were compared with the multi-path particle dosimetry (MPPD) model predictions, which showed that the model overestimated particle deposition in certain lung regions. CONCLUSIONS: Our findings revealed that the particle deposition varied between different little cigar products. The results demonstrated a heterogenous deposition pattern, with higher particle deposition observed in the left and caudal lobes, especially with the mentholated little cigars. Additionally, we identified disparities between our measurements and the MPPD model. This discrepancy highlights the need to enhance the accuracy of models before extrapolating animal study results to human lung deposition. Overall, our study provides valuable insights for estimating the dose of little cigars during smoking for toxicity research.

Evaluation of Six Aromatic Amines in the Mainstream Smoke of Commercial Cigars.

Aromatic amines are a class of carcinogenic compounds present in tobacco smoke that are listed on the U.S. Food and Drug Administration (FDA) list of harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco smoke. The yields of six aromatic amines (1-aminonaphthalene [1-AN], 2-aminonaphthalene [2-AN], 3-aminobiphenyl [3-ABP], 4-aminobiphenyl [4-ABP], ortho-toluidine [o-TOL], and o-anisidine [o-ANI]) in the mainstream smoke from 23 commercial filtered cigars, 16 cigarillos, and 11 large cigars were determined using solid-phase microextraction coupled to gas chromatography triple quadrupole mass spectrometry (SPME headspace GC-MS/MS). The commercial cigars were smoked under the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Recommended Method 64 using a linear cigar smoking machine. The aromatic amine yields in the mainstream smoke from 50 commercial cigars show high levels of variation within and between the products. The average yields of the aromatic amines in the filtered cigars, cigarillos, and large cigars were 108, 371, and 623 ng/cigar for o-TOL; 6, 14, and 22 ng/cigar for o-ANI; 65, 114, and 174 ng/cigar for 1-AN; 25, 59, and 87 ng/cigar for 2-AN; 6, 17, and 27 ng/cigar for 3- ABP; and 8, 11, and 17 ng/cigar for 4-ABP, respectively. The relationships between aromatic amines and (1) total particulate matter (TPM), (2) water-soluble proteins, and (3) water-insoluble proteins were evaluated. We found that the aromatic amines showed a good linear response with TPM on a per cigar basis and showed significant positive correlations with proteins. In addition, the water-insoluble proteins make a greater contribution to the formation of aromatic amines compared to the water-soluble proteins.

Synthetic Cooling Agent and Other Flavor Additives in "Non-Menthol" Cigarettes Marketed in California and Massachusetts After Menthol Cigarette Bans.

This study uses a bioassay and chemical analysis to determine the proportion of newly introduced "non-menthol" cigarette brands with sensory cooling effects, cooling agents added, and any other flavor additives after menthol cigarette bans.

A multi-organ, lung-derived inflammatory response following in vitro airway exposure to cigarette smoke and next-generation nicotine delivery products.

Despite increasing use of in vitro models that closely resemble in vivo human biology, their application in understanding downstream effects of airway toxicity, such as inflammation, are at an early stage. In this study, we used various assays to examine the inflammatory response induced in MucilAir™ tissues and A549 cells exposed to three products known to induce toxicity. Reduced barrier integrity was observed in tissues following exposure to each product, with reduced viability and increased cytotoxicity also shown. Similar changes in viability were also observed in A549 cells. Furthermore, whole cigarette smoke (CS) induced downstream phenotypic THP-1 changes and endothelial cell adhesion, an early marker of atherosclerosis. In contrast, exposure to next-generation delivery product (NGP) aerosol did not induce this response. Cytokine, histological and RNA analysis highlighted increased biomarkers linked to inflammatory pathways and immune cell differentiation following exposure to whole cigarette smoke, including GM-CSF, IL-1ß, cleaved caspase-3 and cytochrome P450 enzymes. As a result of similar observations in human airway inflammation, we propose that our exposure platform could act as a representative model for studying such events in vitro. Furthermore, this model could be used to test the inflammatory or anti-inflammatory impact posed by inhaled compounds delivered to the lung.

Effects of heated tobacco product aerosol extracts on DNA methylation and gene transcription in lung epithelial cells.

AIMS: Smoking causes DNA methylation (DNAm) alterations that lead to lung cancer development. Although the use of heated tobacco products (HTPs) has recently increased, their impact on health remains unclear. This study aimed to evaluate the effects of HTPs on DNAm and gene transcription in human lung epithelial cells in vitro. MAIN METHODS: Human lung adenocarcinoma (A549) cells with type II alveolar epithelial characteristics were treated with aerosol extracts of two HTPs or a smoke extract of combustible reference cigarette (RC). Global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels were quantified using dot blot analysis. Furthermore, reduced representation of bisulfite sequencing, DNA microarray, and quantitative PCR analyses were performed to determine CpG methylation and gene transcription changes induced by HTP and RC. KEY FINDINGS: Global 5-mC and 5-hmC levels were decreased by the RC extract but not the HTP extracts. However, an HTP extract altered the CpG methylation pattern, and Gene Ontology enrichment analysis of the differentially methylated regions of the RC and HTP groups showed a similar pattern. The HTP extract affected gene expression, albeit to a lesser extent than the RC extract. In particular, the HTP extract markedly affected the mRNA expression and promoter methylation of cytochrome P450 family 1 subfamily A member 1 (CYP1A1), which is associated with carcinogenic risk. SIGNIFICANCE: The study results suggest that HTPs as well as conventional combustible cigarettes can alter CpG methylation and gene transcription in lung epithelial cells.

Effects of aerosols from heated tobacco products with flavors on the discoloration of bovine tooth enamel.

OBJECTIVES: This in vitro study assessed the potential of tooth discoloration by aerosols generated from three heated tobacco products (HTPs) with different specifications: in-direct heating tobacco system platform 1.0a (IT1.0a), in-direct heating tobacco system platform 2.0a (IT2.0a), and direct heating tobacco system platform 3.0a (DT3.0a). In addition, three flavor types (regular, menthol, and berry menthol) were selected for each HTP to characterize the effect of flavor types on tooth discoloration. MATERIAL AND METHODS: Six bovine tooth samples were exposed directly to aerosols generated from one pack of each HTP: 350 puffs for IT1.0a, 325 puffs for IT2.0a, and 220 puffs for DT3.0a. Six bovine tooth samples were also exposed to air (350 puffs) and smoke generated from one pack of cigarettes (160 puffs) as negative and positive controls, respectively. The color of each tooth sample was measured before and after exposure. The overall color changes were assessed using overall color differences (ΔE) calculated according to the Commission International de I'Eclairage color system. A one-way analysis of variance followed by Tukey's post hoc test was used to compare ΔE among bovine tooth samples exposed to air, cigarette smoke, and aerosols generated from each HTP. RESULTS: ΔE values for tooth samples exposed to air and aerosols generated from the three HTPs (IT1.0a, IT2.0a, and DT3.0a) were significantly lower than ΔE value for tooth samples exposed to cigarette smoke. ΔE values obtained with DT3.0a were significantly higher than those obtained with air-exposed control samples. However, ΔE values obtained with IT1.0a and IT2.0a were not significantly different from that obtained with air-exposed control samples. No HTPs showed significant differences in ΔE values among the three flavor types. CONCLUSIONS: This study showed that HTP aerosols reduce tooth discoloration potential compared with cigarette smoke, regardless of flavor types, and the tooth discoloration potential of the product may depend on product specifications.

Study on the effect of enzymatic treatment of tobacco on HnB cigarettes and microbial succession during fermentation.

Starch and cellulose are the fundamental components of tobacco, while their excessive content will affect the quality of tobacco. Enzymatic treatment with different enzymes is a promising method to modulate the chemical composition and improve the sensory quality of tobacco leaves. In this study, enzymatic treatments, such as amylase, cellulase, and their mixed enzymes, were used to improve tobacco quality, which could alter the content of total sugar, reducing sugar, starch, and cellulose in tobacco leaves. The amylase treatment changed surface structure of tobacco leaves, increased the content of neophytadiene in tobacco by 16.48%, and improved the total smoking score of heat-not-burn (HnB) cigarette products by 5.0 points compared with the control. The Bacillus, Rubrobacter, Brevundimonas, Methylobacterium, Stenotrophomonas, Acinetobacter, Pseudosagedia-chlorotica, and Sclerophora-peronella were found to be significant biomarkers in the fermentation process by LEfSe analysis. The Basidiomycota and Agaricomycetes were significantly correlated with aroma and flavor, taste, and total score of HnB. The results showed that microbial community succession occurred due to amylase treatment, which promoted the formation of aroma compounds, and regulated the chemical composition of tobacco, and improved tobacco quality during tobacco fermentation. This study provides a method for enzymatic treatment to upgrade the quality of tobacco raw materials, thereby improving the quality of HnB cigarettes, and the potential mechanism is also revealed by chemical composition and microbial community analysis. KEY POINTS: Enzymatic treatment can change the chemical composition of tobacco leaves. The microbial community was significantly affected by enzymatic treatment. The quality of HnB cigarettes was significantly improved by amylase treatment.

Influence of Tobacco Variety and Curing on Free Radical Production in Cigarette Smoke.

INTRODUCTION: Cigarette smoke contains highly reactive free radicals thought to play an important role in tobacco smoke-induced harm. Previously, large variations in free radical and toxicant output have been observed in commercial cigarettes. These variations are likely because of cigarette design features (paper, filter, and additives), tobacco variety (burley, bright, oriental, etc.), and tobacco curing methods (air, sun, flue, and fire). Previous reports show that tobacco varieties and curing methods influence the production of tobacco smoke constituents like the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK). AIMS AND METHODS: We evaluated free radical, nicotine, and NNK production in cigarette smoke from cigarettes produced with 15 different types of tobacco. Gas-phase free radicals were captured by spin trapping with N-tert-butyl-α-phenylnitrone and particulate-phase radicals were captured on a Cambridge Filter pad (CFP). Both types of radicals were analyzed using electron paramagnetic resonance spectroscopy. Nicotine and NNK were extracted from the CFP and analyzed by gas chromatography flame ionization detection and liquid chromatography-mass spectrometry, respectively. RESULTS: Gas-phase radicals varied nearly 8-fold among tobacco types with Saint James Perique tobacco producing the highest levels (42 ±â€…7 nmol/g) and Canadian Virginia tobacco-producing the lowest levels (5 ±â€…2 nmol/g). Nicotine and NNK levels in smoke varied 14-fold and 192-fold, respectively, by type. Gas-phase free radicals were highly correlated with NNK levels (r = 0.92, p < .0001) and appeared to be most impacted by tobacco curing method. CONCLUSIONS: Altogether, these data suggest that tobacco types used in cigarette production may serve as a target for regulation to reduce harm from cigarette smoking. IMPLICATIONS: Variations in cigarette free radical and NNK levels vary based on the tobacco variety and curing method. Reducing the ratio of high-producing free radical and NNK tobacco types offer a potential tool for regulators and producers looking to reduce toxicant output from cigarettes.

Carbonyl Yields in Cigars under Three Smoking Regimens Using a Linear Smoking Machine.

This study used standard linear smoking machines and puffing protocols to generate data on carbonyl yields in mainstream smoke from 11 unfiltered sheet-wrapped cigars (SWC), seven leaf-wrapped cigars (LWC), and two Kentucky reference cigarettes (3R4F, 1R6F). Carbonyl yields in cigar and cigarette products were determined using three different smoking regimens: International Organization for Standardization (ISO), Canadian Intense (CI), and Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Recommended Method (CRM) No. 64 (CRM64, Routine Analytical Cigar-Smoking Machine─Specifications, Definitions and Standard Conditions). Mainstream tobacco smoke was collected using a smoking machine fitted with an impinger containing 2,4-dinitrophenylhydrazine (DNPH) and carbonyl compounds quantified using liquid chromatography with an ultraviolet detector. Commercial SWC and LWC generated comparable formaldehyde yields (SWC, 9.4-28 µg/cigar [ISO], 8.2-43 µg/cigar [CI], 8.6-13 µg/cigar [CRM64]; LWC, 11-13 µg/cigar [ISO], 11-22 µg/cigar [CI], 16-21 µg/cigar [CRM64]) and acrolein yields; however, LWC generated higher acetaldehyde yields compared to SWC, using CI and CRM64 regimens. Reference cigarettes using standard puffing regimens generated carbonyl yields within reported ranges and 5-10% RSDs, whereas the CRM64 regimen generated lower carbonyl yields and 12-14% RSDs. Reference cigarettes generated higher formaldehyde yields using cigarette smoking regimens (21-28 µg/cigarette under ISO, 76-96 µg/cigarette under CI) but comparable formaldehyde yields under CRM64 (12-14 µg/cigarette). In addition, this study evaluated physical parameters (e.g., tobacco weight, length, diameter, circumference, tobacco rod density) that show the correlation between tobacco weight, tobacco rod density, and acetaldehyde yields under the three smoking regimens. Carbonyl yields in the mainstream smoke of cigar products using the three smoking regimens were highly variable; however, the CI smoking regimen may provide meaningful analytical information regarding cigar smoke constituents, with lower likelihood of self-extinguishment due to the short puffing intervals.

Conventional tobacco products harbor unique and heterogenous microbiomes.

While an increasing number of studies have evaluated tobacco microbiomes, comparative microbiome analyses across diverse tobacco products are non-existent. Moreover, to our knowledge, no previous studies have characterized the metabolically-active (live) fraction of tobacco bacterial communities and compared them across products. To address these knowledge gaps, we compared bacterial communities across four commercial products (cigarettes, little cigars, cigarillos and hookah) and one research cigarette product. After total DNA extraction (n = 414) from all samples, the V3V4 region of the 16S rRNA gene was sequenced on the Illumina HiSeq platform. To identify metabolically-active bacterial communities within these products, we applied a coupled 5-bromo-2'-deoxyuridine labeling and sequencing approach to a subset of samples (n = 56). Each tobacco product was characterized by its signature microbiome, along with a shared microbiome across all tobacco products consisting of Pseudomonas aeruginosa, P. putida, P. alcaligenes, Bacillus subtilis, and Klebsiella pneumoniae. Comparing across products (using Linear discriminant analysis Effect Size (LEfSe)), a significantly higher (p < 0.05) relative abundance of Klebsiella and Acinetobacter was observed in commercial cigarettes, while a higher relative abundance of Pseudomonas and Pantoea was observed in research cigarettes. Methylorubrum and Paenibacillus were higher in hookah, and Brevibacillus, Lactobacillus, Bacillus, Lysinibacillus, and Staphylococcus were higher in little cigars and cigarillos. Across all products, the majority of the metabolically-active bacterial communities belonged to the genus Pseudomonas, followed by several genera within the Firmicutes phylum (Bacillus, Terribacillus, and Oceanobacillus). Identification of some metabolically-active pathogens such as Bacillus cereus and Haemophilus parainfluenzae in commercial products is of concern because of the potential for these microorganisms to be transferred to users' respiratory tracts via mainstream smoke. Future work is warranted to evaluate the potential impact of these tobacco bacterial communities on users' oral and lung microbiomes, which play such an important role on the spectrum from health to disease.

A Method to Amend Cigarettes With Menthol for Use in Research.

INTRODUCTION: Studies have evaluated the role of menthol cigarettes on various addiction-related outcomes; however, the effect of varying menthol content on these outcomes has not been evaluated. We developed a method to amend non-menthol SPECTRUM Research Cigarettes to contain menthol at four different levels. AIMS AND METHODS: SPECTRUM Research Cigarettes, NRC 600 (0.8 mg nicotine; 10 mg tar), were modified to contain target menthol amounts at 3, 6, and 12 mg/cigarette by injecting 25 µL ethanol/triacetin/menthol solutions of varying concentrations (120 mg menthol/mL, 240 mg/mL, and 480 mg/mL) into four distinct locations in the filter and tobacco rod. Menthol content was tested in triplicate in the whole cigarette and in the tobacco rod and filter at 1, 24, 48, and 72 hours for each target menthol level using an extraction solution of quinoline in methyl-tert-butyl ether and measured using gas chromatography with flame ionization detection. RESULTS: Injections into the filter and tobacco rod (12.5 µL each) yielded equal menthol distribution up to 72 hours. However, total menthol content decreased from an average of 90.3% of the target menthol concentration at 1 hour to 80.7% at 72 hours in cigarettes stored individually in glass tubes at room temperature. Analysis of urinary menthol glucuronide confirmed that amended cigarettes used within 24 hours of injection delivered dose-related menthol levels to participants in a clinical laboratory setting. CONCLUSION: This method can be used to modify cigarettes with a range of reliable menthol levels in both filter and tobacco rod for use in laboratory and clinical research. IMPLICATIONS: This study presents a technique for modifying cigarettes with different levels of menthol that can reliably deliver dose-related menthol levels to participants when smoked in a clinical study. The technique can be used to quickly amend cigarettes to examine the independent effects of varying flavor and additive levels on smoking behavior, nicotine pharmacokinetics, mainstream smoke emissions, and other laboratory or clinical research outcomes.

Mouth Level Intake of Nicotine from Three Brands of Little Filtered Cigars with Widely Differing Product Characteristics Among Adult Consumers.

Little filtered cigars are tobacco products with many cigarette-like characteristics. However, despite cigars falling under the U.S. Food and Drug Administration regulatory authority, characterizing flavors, which are still allowed in little filtered cigars, and filter design may influence how people use the products and the resulting exposure to harmful and potentially harmful constituents. We estimated nicotine mouth level intake (MLI) from analyses of little cigar filter butt solanesol levels, brand characteristics, carbon monoxide boost, and puff volume in 48 dual cigarette/cigar users during two repeat bouts of ad lib smoking of three little filtered cigar brands. Mean nicotine MLI for the three brands was significantly different with Swisher Sweets (0.1% ventilation) Cherry at 1.20 mg nicotine, Cheyenne Menthol (1.5%) at 0.63 mg, and Santa Fe unflavored (49%) at 0.94 mg. The association between nicotine MLI and puff volume was the same between Cheyenne Menthol and Santa Fe unflavored. However, these were different from Swisher Sweets Cherry. At least five main factors─flavor, ventilation, filter design, nicotine delivery related to tar, and user puff volume─may directly or indirectly impact MLI and its association with other measures. We found that users of little filtered cigars that have different filter ventilation and flavor draw dissimilar amounts of nicotine from the product, which may be accompanied by differences in exposure to other harmful smoke constituents.

Flavours and flavourings in waterpipe products: a comparison between tobacco, herbal molasses and steam stones.

ObjectivesFlavoured products are especially appealing to youth and contribute to the onset of waterpipe smoking and continued use of waterpipe tobacco. The goal of database and chemical analysis was to provide a clear overview of commonly used flavours and flavourings in tobacco and related waterpipe products, that is, herbal molasses and steam stones. METHODS: In 2019, 249 waterpipe tobacco products were registered in the European Common Entry Gate by manufacturers to be marketed in The Netherlands. Flavour categories were assigned to the registered products based on their brand names and product descriptions. Nicotine and eleven 1111 flavourings were identified and quantified in waterpipe tobacco (n=8), herbal molasses (n=7) and steam stones (n=4) by extraction and gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: Flavour categories could be assigned to 237 of 249 registered waterpipe tobacco products. Eight flavour main categories and 48 unique subcategories were identified and presented in a flavour wheel. All registered waterpipe tobacco products were flavoured, and the majority (78%) was fruit flavoured. Herbal molasses contained similar median flavouring levels, and steam stones contained lower median levels compared with waterpipe tobacco. Flavourings in waterpipe products were almost exclusively fruity and sweet, often in combination with menthol/mint flavourings. CONCLUSIONS: This study is the first to present a waterpipe tobacco flavour wheel, providing a quick overview of waterpipe tobacco flavours and thereby aiding communication among experts around the globe. GC-MS analysis revealed that the most prevalent flavourings are present in similar levels in herbal and tobacco waterpipe products. Banning flavourings in all waterpipe products would be a good strategy to reduce waterpipe smoking among youth.

Variability in Urinary Nicotine Exposure Biomarker Levels Between Waves 1 (2013-2014) and 2 (2014-2015) in the Population Assessment of Tobacco and Health Study.

INTRODUCTION: To date, no studies have evaluated the consistency of biomarker levels in people who smoke over a long-time period in real-world conditions with a large number of subjects and included use behavior and measures of nicotine metabolism. We evaluated the variability of biomarkers of nicotine exposure over approximately a 1-year period in people who exclusively smoke cigarettes, including intensity and recency of use and brand switching to assess impact on understanding associations with product characteristics. AIMS AND METHODS: Multivariate regression analysis of longitudinal repeated measures of urinary biomarkers of nicotine exposure from 916 adults in the Population Assessment of Tobacco and Health (PATH) Study with demographic characteristics and use behavior variables. Intraclass correlation coefficients (ICCs) were calculated to examine individual variation of nicotine biomarkers and the uncertainty of repeat measures at two time points (Waves 1 and 2). RESULTS: Age, race, and urinary creatinine were significant covariates of urinary cotinine. When including use behavior, recency, and intensity of use were highly significant and variance decreased to a higher extent between than within subjects. The ICC for urinary cotinine decreased from 0.7530 with no use behavior variables in the model to 0.5763 when included. Similar results were found for total nicotine equivalents. CONCLUSIONS: Urinary nicotine biomarkers in the PATH Study showed good consistency between Waves 1 and 2. Use behavior measures such as time since last smoked a cigarette and number of cigarettes smoked in the past 30 days are important to include when assessing factors that may influence biomarker concentrations. IMPLICATIONS: The results of this study show that the consistency of the nicotine biomarkers cotinine and total nicotine equivalents in spot urine samples from Waves 1 to 2 of the PATH Study is high enough that these data are useful to evaluate the association of cigarette characteristics with biomarkers of exposure under real-world use conditions.

Chemical analysis of selected harmful and potentially harmful constituents and in vitro toxicological evaluation of leading flavoured e-cigarette aerosols in the Chinese market.

Use of electronic cigarettes (e-cigarettes) has increased significantly over the past decade due to consumer perception that these products represent a less risky alternative to combustible cigarettes. E-liquids generally contain a simple mix of vegetable glycerin, propylene glycerol, nicotine, organic acids, and flavourings. Regulators require that harmful and potentially harmful constituents (HPHCs) that might cause harm to the consumer must be monitored in the aerosol generated by e-cigarettes and in cigarette smoke (CS). To quantify HPHCs in aerosols from commercial flavoured e-cigarettes in Chinese market, this study has systematically compared levels of HPHCs, including eight carbonyls, five volatile organic compounds, four tobacco-specific nitrosamines, 16 polycyclic aromatic hydrocarbons, and seven heavy metals, in the aerosols of four market-leading flavoured e-cigarettes and mainstream CS, alongside in vitro cytotoxicity and mutagenicity assays. The vast majority of HPHCs were either undetected or significantly lower in the e-cigarette aerosols than in commercial CS or reference CS (3R4F). Where HPHCs were detected, there were small variations among the different flavoured e-cigarettes. In the neutral red uptake and Ames assays, aqueous extracts of the e-cigarette aerosols did not induce obvious cytotoxicity or mutagenicity, whereas CS aqueous extract showed dose-related cytotoxicity and mutagenicity. Collectively, these results indicate that use of e-cigarettes might potentially lead to a significant reduction in exposure to harmful substances, with fewer cytotoxic and mutagenic effects, as compared with conventional smoking. Further studies based on human puffing conditions and longer evaluation periods will be needed to substantiate this potential.

Rapid and sensitive HPLC-MS/MS method for the quantification of dopamine, GABA, serotonin, glutamine and glutamate in rat brain regions after exposure to tobacco cigarettes.

Tobacco smoking is a preventable main cause of fatal diseases. Accurate measurements of the effects it has on neurotransmitters are essential in developing new strategies for smoking cessation. Moreover, measurements of neurotransmitter levels can aid in developing drugs that counteract the effects of smoking. The aim of this study is to develop and validate a fast, simultaneous and sensitive method for measuring the levels of neurotransmitters in rat brain after the exposure of tobacco cigarettes. The selected neurotransmitters include dopamine, GABA, serotonin, glutamine and glutamate. The method is based on high-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved within 3 min using a Zorbax SB C18 column (3.0 × 100 mm, 1.8 µm particle size). The mobile phase consisted of HPLC-grade water and acetonitrile each containing 0.3% heptafluorobutyric acid and 0.5% formic acid at gradient conditions. The linear range was 0.015-0.07, 825-7,218, 140-520, 63.42-160.75 and 38.25 × 103 to 110.35 × 103  ng/ml for dopamine, GABA, serotonin, glutamine and glutamate, respectively. Inter- and intra-run accuracy were in the range 97.82-103.37% with a precision (CV%) of ≤0.90%. The results revealed that 4 weeks of cigarette exposure significantly increased neurotransmitter levels after exposure to tobacco cigarettes in various brain regions, including the hippocampus and the amygdala. This increase in neurotransmitters levels may in turn activate the nicotine dependence pathway.