Canadian Intense Research Articles

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.

Hydrogen Cyanide and Aromatic Amine Yields in the Mainstream Smoke of 60 Little Cigars.

Mainstream smoke yields of hydrogen cyanide (HCN) and three aromatic amines, 1-aminonaphthalene, 2-aminonaphthalene, and 4-aminobiphenyl, from 60 little cigar brands currently on the US market were measured for both International Organization for Standardization (ISO) and Canadian Intense (CI) smoking regimens. The smoke yields are compared with those from 50 cigarette products measured by Counts et al. of Philip Morris USA (PMUSA) in 2005 [Counts et al. Regul. Toxicol. Pharmacol. 2005 41, 185-227] and 50 cigarette products measured by the Centers for Disease Control and Prevention (CDC) in cooperation with the Food and Drug Administration (FDA) in 2012 [Tynan et al. Consumption of Cigarettes and Combustible Tobacco: United States, 2000-2011. In Morbidity and Mortality Weekly Report; Centers for Disease Control and Prevention, 2012; 565-580]. For the little cigars, the average HCN yield with the ISO smoking regimen is 335 μg/cigar (range: 77-809 μg/cigar), which is 332% higher than the average of 50 PMUSA 2005 cigarettes and 243% higher than the average of 50 CDC/FDA 2012 cigarettes. For the CI smoking regimen, the average HCN yield is 619 μg/cigar (range: 464-1045 μg/cigar), which is 70.5% higher than the average of 50 PMUSA 2005 cigarettes and 69% higher than the average of the 50 CDC/FDA 2012 cigarettes. For aromatic amines, the average ISO smoking regimen smoke yields are 36.6 ng/cigar (range: 15.9-70.6 ng/cigar) for 1-aminonaphthalene, 24.6 ng/cigar (range: 12.3-36.7 ng/cigar) for 2-aminonaphthalene, and 5.6 ng/cigar (range: 2.3-17.2 ng/cigar) for 4-aminobiphenyl. The average ISO yields of aromatic amines from little cigars are 141% to 210% higher compared to the average yields of 50 PMUSA cigarettes. The average CI smoke regimen yields are 73.0 ng/cigar (range: 32.1-112.2 ng/cigar) for 1-aminonaphthalene, 45.2 ng/cigar (range: 24.6-74.8 ng/cigar) for 2-aminonaphthalene, and 12.7 ng/cigar (range: 5.5-37.5 ng/cigar) for 4-aminobiphenyl. The average CI aromatic amine yields are 143% to 220% higher compared to the average yields of 50 PMUSA cigarettes, almost identical to the relative yields under the ISO smoking regimen. Both HCN and aromatic amine yields are 1.5× to 3× higher for the tested little cigars than for the conventional cigarettes; however, there are notable differences in the relationships of these yields to certain product characteristics, such as weight, ventilation, and tobacco type. The higher smoke yields of these compounds from little cigars indicates that cigar smokers may be at risk of a higher exposure to HCN and aromatic amines on a per stick basis and thus increased health concerns.

Volatile Organic Compounds in Mainstream Smoke of Sixty Domestic Little Cigar Products.

The mainstream smoke yields of five volatile organic compounds (VOCs) were determined from 60 commercial U.S. little cigar products under ISO 3308 and Canadian Intense (CI) smoking regimens on linear smoking machines using a gas sampling bag collection. The five VOCs, 1,3-butadiene, acrylonitrile, benzene, isoprene, and toluene were analyzed using an automated GC/MS analytical method validated for measuring various VOCs in mainstream smoke. The VOCs range in amounts from micrograms to milligrams per little cigar. VOC deliveries vary considerably among the little cigar products under the ISO smoking regimen primarily due to varying filter ventilation. Under the CI smoking regimen where filter ventilation is blocked, the delivery range narrows, although individual and total VOC yields are approximately 2 fold higher than those under the ISO smoking regimen. Correlation analysis reveals strong associations between acrylonitrile and 1,3-butadiene or toluene under the ISO smoking regimen. Compared to cigarettes, little cigars delivered substantially higher VOC mainstream smoke yields under both ISO and CI smoking regimens. Moreover, little cigar smoke also contains higher VOCs than cigarette smoke when adjusted for mass of tobacco.

Free Radical and Nicotine Yields in Mainstream Smoke of Chinese Marketed Cigarettes: Variation with Smoking Regimens and Cigarette Brands.

Free radicals and nicotine are components of cigarette smoke that are thought to contribute to the development of smoking-induced diseases. China has the largest number of smokers in the world, yet little is known about the yields of tobacco smoke constituents in different Chinese brands of cigarettes. In this study, gas-phase and particulate-phase free radicals as well as nicotine yields were quantified in mainstream cigarette smoke from five popular Chinese brands and two research cigarettes (3R4F and 1R6F). Mainstream smoke was generated under International Organization of Standardization (ISO) and Canadian Intense (CI) smoking regimens using a linear smoking machine. Levels of free radicals and nicotine were measured by electron paramagnetic resonance spectroscopy (EPR) and gas chromatography with flame-ionization detection, respectively. Under the ISO puffing regimen, Chinese brand cigarettes produced an average of 3.0 ± 1.2 nmol/cig gas-phase radicals, 118 ± 44.7 pmol/cig particulate-phase radicals, and 0.6 ± 0.2 mg/cig nicotine. Under the CI puffing regimen, Chinese brand cigarettes produced an average of 5.6 ± 1.2 nmol/cig gas-phase radicals, 282 ± 92.1 pmol/cig particulate-phase radicals, and 2.1 ± 0.4 mg/cig nicotine. Overall, both gas- and particulate-phase free radicals were substantially lower compared to the research cigarettes under both regimens, whereas no significant differences were observed for nicotine levels. When Chinese brands were compared, the highest free radical and nicotine yields were found in "LL" and "BS" brands, while lowest levels were found in "YY". These results suggested that the lower radical delivery by Chinese cigarettes compared to United States reference cigarettes may be associated with reductions in oxidant-related harm.

Cigarette whole smoke solutions disturb mucin homeostasis in a human in vitro airway tissue model

Many cigarette smoke-associated airway diseases involve alterations in mucin homeostasis. With the rationale that relevant tissue responses can be measured to evaluate the adverse health effects of tobacco products, we assessed changes in mucin secretion and the density and size of goblet cells in an in vitro human air-liquid-interface (ALI) airway tissue model after exposure to a tobacco smoke solution. Cultures were exposed daily for up to five consecutive days to a whole smoke solution (WSS) prepared by machine smoking Marlboro Red or Marlboro Silver cigarettes using the Canadian Intense (CI) protocol. Both WSSs induced concentration- and time-related hypersecretion of mucins 5AC and 5B, accompanied by up-regulation of the respective mucin genes. Mucin secretion returned to baseline levels following a 14-day recovery period. Mucin-secreting goblet cells exhibited increased cell density and decreased size after 5 daily treatments then recovered to their normal size, but with decreased cell density, 14 days after the last treatment. The beating frequency of ciliated cells, which plays a key role in mucociliary clearance, was increased by 5 daily treatments with the CI WSSs then reverted to baseline levels following a 7-day recovery. Taken together, our results indicate that ALI cultures can be used to measure the modulation of mucin production, secretion, and clearance, disturbances that are manifested in tobacco smoke-related diseases, such as chronic obstructive pulmonary disease. Measuring tissue responses directly relevant to the respiratory toxicity of cigarette smoke may provide useful information in support of science-based regulatory decisions.

Influence of Smoking Puff Parameters and Tobacco Varieties on Free Radicals Yields in Cigarette Mainstream Smoke.

Cigarette smoke is a major exogenous source of free radicals, and the resulting oxidative stress is one of the major causes of smoking-caused diseases. Yet, many of the factors that impact free radical delivery from cigarettes remain unclear. In this study, we machine-smoked cigarettes and measured the levels of gas- and particulate-phase radicals by electron paramagnetic resonance (EPR) spectroscopy using standardized smoking regimens (International Organization of Standardization (ISO) and Canadian Intense (CI)), puffing parameters, and tobacco blends. Radical delivery per cigarette was significantly greater in both gas (4-fold) and particulate (6-fold) phases when cigarettes were smoked under the CI protocol compared to the ISO protocol. Total puff volume per cigarette was the major factor with radical production being proportional to total volume, regardless of whether volume differences were achieved by changes in individual puff volume or puff frequency. Changing puff shape (bell vs sharp vs square) or puff duration (1-5 s), without changing volume, had no effect on radical yields. Tobacco variety did have a significant impact on free radical production, with gas-phase radicals highest in reconstituted > burley > oriental > bright tobacco and particulate-phase radicals highest in burley > bright > oriental > reconstituted tobacco. Our findings show that modifiable cigarette design features and measurable user smoking behaviors are key factors determining free radical exposure in smokers.

A Survey of Nicotine Yields in Small Cigar Smoke: Influence of Cigar Design and Smoking Regimens.

Although the popularity of small cigar brands that resemble cigarettes, including both little cigars (LC) and filtered cigars (FC), has been on the rise, little is known about the delivery of nicotine from these products. Our objective was to determine the nicotine yields of small cigars in comparison to cigarettes. Nicotine yields from LC, FC, and 3R4F and 1R6F research cigarettes were determined from mainstream smoke generated on a smoking machine under the International Organization of Standardization (ISO) and Canadian Intense (CI) methods. Market characteristics (price and package label) and physical features (filter ventilation, product weight and filter weight, product length, and diameter) were also determined for eight brands of small cigars. Nicotine yields in small cigars averaged 1.24 and 3.49 mg/unit on ISO and CI regimens, respectively, compared with 0.73 and 2.35 mg/unit, respectively, for the research cigarettes. Nicotine yields per puff were similar between small cigars and cigarettes. We also found that FC did not differ from LC in nicotine yields. FC and LC differ from each other in many physical design features (unit weight, filter weight, and filter length), but are similar in others (unit length, diameter, and filter ventilation). Nicotine delivery from small cigars is similar to or greater than that from cigarettes. Thus, for future research and regulatory purposes, standard definitions need to be developed for small cigars, and FC and LC should be evaluated as separate entities. Small cigars are similar to cigarettes in their design and use. Although nicotine yields per puff were similar between products, small cigars delivered substantially higher amounts of nicotine per unit than cigarettes. These findings support the growing body of evidence to justify regulating all small cigars, including LC and FC in a similar fashion as cigarettes.

Simultaneous Determination of Furan and Vinyl Acetate in Vapor Phase of Mainstream Cigarette Smoke by GC-MS.

A simple and sensitive method for simultaneous determination of furan and vinyl acetate (VA) in vapor phase of mainstream cigarette smoke with cold trap and gas chromatography-mass spectrometry (GC-MS) was developed. A Cambridge filter pad (CFP) was placed in front of the impingers of smoking machine to remove the particle phase from cigarette smoke. Furan and VA in vapor phase of mainstream cigarette smoke were collected in two impingers connected in series by filled with methanol at -78°C. The solutions were added with deuterium-labeled furan-d4 and VA-d6 as internal standards and analyzed by GC-MS. The results showed that the calibration curves for furan and VA were linear (r2 > 0.9995) over the studied concentration range. The intra- and inter-day precision values for furan and VA were <7.07% and <9.62%, respectively. The extraction recoveries of furan and VA were in the range of 94.5-97.7% and 92.3-94.9%, respectively. Moreover, the limits of detection for furan and VA were 0.028 µg mL-1 and 1.3 ng mL-1, respectively. The validated method has been successfully applied to determine the emissions of furan and VA in the vapor phase of mainstream cigarette smoke under International Organization for Standardization (ISO) and Canadian Intense (CI) smoking regimen.

Tobacco-Specific Nitrosamines in the Tobacco and Mainstream Smoke of U.S. Commercial Cigarettes.

Tobacco-specific nitrosamines (TSNAs) are N-nitroso-derivatives of pyridine-alkaloids (e.g., nicotine) present in tobacco and cigarette smoke. Two TSNAs, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are included on the Food and Drug Administration's list of harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco. The amounts of four TSNAs (NNK, NNN, N-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT)) in the tobacco and mainstream smoke from 50 U.S. commercial cigarette brands were measured from November 15, 2011 to January 4, 2012 using a validated HPLC/MS/MS method. Smoke samples were generated using the International Organization of Standardization (ISO) and Canadian Intense (CI) machine-smoking regimens. NNN and NAT were the most abundant TSNAs in tobacco filler and smoke across all cigarette brands, whereas NNK and NAB were present in lesser amounts. The average ratios for each TSNA in mainstream smoke to filler content is 29% by the CI smoking regimen and 13% for the ISO machine-smoking regimen. The reliability of individual TSNAs to predict total TSNA amounts in the filler and smoke was examined. NNN, NAT, and NAB have a moderate to high correlation (R2 = 0.61-0.98, p < 0.0001), and all three TSNAs individually predict total TSNAs with minimal difference between measured and predicted total TSNA amounts (error < 7.4%). NNK has weaker correlation (R2 = 0.56-0.82; p < 0.0001) and is a less reliable predictor of total TSNA quantities. Tobacco weight and levels of TSNAs in filler influence TSNA levels in smoke from the CI machine-smoking regimen. In contrast, filter ventilation is a major determinant of levels of TSNAs in smoke by the ISO machine-smoking regimen. Comparative analysis demonstrates substantial variability in TSNA amounts in tobacco filler and mainstream smoke yields under ISO and CI machine-smoking regimens among U.S. commercial cigarette brands.

Multivariate Statistical Analysis of Cigarette Design Feature Influence on ISO TNCO Yields.

The aim of this study is to explore how differences in cigarette physical design parameters influence tar, nicotine, and carbon monoxide (TNCO) yields in mainstream smoke (MSS) using the International Organization of Standardization (ISO) smoking regimen. Standardized smoking methods were used to evaluate 50 U.S. domestic brand cigarettes and a reference cigarette representing a range of TNCO yields in MSS collected from linear smoking machines using a nonintense smoking regimen. Multivariate statistical methods were used to form clusters of cigarettes based on their ISO TNCO yields and then to explore the relationship between the ISO generated TNCO yields and the nine cigarette physical design parameters between and within each cluster simultaneously. The ISO generated TNCO yields in MSS are 1.1-17.0 mg tar/cigarette, 0.1-2.2 mg nicotine/cigarette, and 1.6-17.3 mg CO/cigarette. Cluster analysis divided the 51 cigarettes into five discrete clusters based on their ISO TNCO yields. No one physical parameter dominated across all clusters. Predicting ISO machine generated TNCO yields based on these nine physical design parameters is complex due to the correlation among and between the nine physical design parameters and TNCO yields. From these analyses, it is estimated that approximately 20% of the variability in the ISO generated TNCO yields comes from other parameters (e.g., filter material, filter type, inclusion of expanded or reconstituted tobacco, and tobacco blend composition, along with differences in tobacco leaf origin and stalk positions and added ingredients). A future article will examine the influence of these physical design parameters on TNCO yields under a Canadian Intense (CI) smoking regimen. Together, these papers will provide a more robust picture of the design features that contribute to TNCO exposure across the range of real world smoking patterns.

Mainstream Smoke Levels of Volatile Organic Compounds in 50 U.S. Domestic Cigarette Brands Smoked With the ISO and Canadian Intense Protocols.

A significant portion of the increased risk of cancer and respiratory disease from exposure to cigarette smoke is attributed to volatile organic compounds (VOCs). In this study, 21 VOCs were quantified in mainstream cigarette smoke from 50U.S. domestic brand varieties that included high market share brands and 2 Kentucky research cigarettes (3R4F and 1R5F). Mainstream smoke was generated under ISO 3308 and Canadian Intense (CI) smoking protocols with linear smoking machines with a gas sampling bag collection followed by solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) analysis. For both protocols, mainstream smoke VOC amounts among the different brand varieties were strongly correlated between the majority of the analytes. Overall, Pearson correlation (r) ranged from 0.68 to 0.99 for ISO and 0.36 to 0.95 for CI. However, monoaromatic compounds were found to increase disproportionately compared to unsaturated, nitro, and carbonyl compounds under the CI smoking protocol where filter ventilation is blocked. Overall, machine generated "vapor phase" amounts (µg/cigarette) are primarily attributed to smoking protocol (e.g., blocking of vent holes, puff volume, and puff duration) and filter ventilation. A possible cause for the disproportionate increase in monoaromatic compounds could be increased pyrolysis under low oxygen conditions associated with the CI protocol. This is the most comprehensive assessment of volatile organic compounds (VOCs) in cigarette smoke to date, encompassing 21 toxic VOCs, 50 different cigarette brand varieties, and 2 different machine smoking protocols (ISO and CI). For most analytes relative proportions remain consistent among U.S. cigarette brand varieties regardless of smoking protocol, however the CI smoking protocol did cause up to a factor of 6 increase in the proportion of monoaromatic compounds. This study serves as a basis to assess VOC exposure as cigarette smoke is a principle source of overall population-level VOC exposure in the United States.

Mouth Level Exposure and Similarity to Machine-smoked Constituent Yields.

The goal of this review was to evaluate which standard machine-smoking regimen may be most appropriate to inform tobacco product regulation based on the fraction of cigarette smoke yields that best represents the range of human smoke exposures. We searched PubMed and Web of Science to identify peer-reviewed studies that reported percentages of smokers who smoked more or less like a particular machine-smoking regimen based on human mouth level exposure (MLE) tobacco constituent yields. Three studies met our inclusion criteria and were reviewed. Studies found that Canadian Intense (CI) yields were equal to or greater than 86% to 97% of smokers' nicotine and tar MLE yields. MLE yields indicate that a small percentage of individuals (less than 14%) are exposed to nicotine and tar yields equal to or greater than those measured by the CI regimen. Whereas no machine-smoking regimen reflects human puffing behavior with complete accuracy, based on MLE data, CI constituent yields constitute the best representation of exposure that encompasses the majority of smokers, and may be the most informative for regulatory purposes.

Updates of CORESTA Recommended Methods after Further Collaborative Studies Carried Out under Both ISO and Health Canada Intense Smoking Regimes

Abstract During 2012, three CORESTA Recommended Methods (CRMs) (1-3) were updated to include smoke yield and variability data under both ISO (4) and the Canadian Intense (CI) (5) smoking regimes. At that time, repeatability and reproducibility data under the CI regime on smoke analytes other than “tar”, nicotine and carbon monoxide (6) and tobacco-specific nitrosamines (TSNAs) (7) were not available in the public literature. The subsequent work involved the determination of the mainstream smoke yields of benzo[a]-pyrene, selected volatiles (benzene, toluene, 1,3-butadiene, isoprene, acrylonitrile), and selected carbonyls (acetaldehyde, formaldehyde, propionaldehyde, butyraldehyde, crotonaldehyde, acrolein, acetone and 2-butanone) in ten cigarette products followed by statistical analyses according to the ISO protocol (8). This paper provides some additional perspective on the data variability under the ISO and CI smoking regimes not given in the CRMs.

Aspects of the design protocol and the statistical methods for analysis of tar, nicotine and carbon monoxide yields in cigarette smoke that can affect the measurement variability within collaborative studies

Statistical principles described in ISO 5725–1 (1994) are a robust basis for evaluating cigarette smoke data from collaborative studies under the ISO 3308 machine smoking and for specifying the criteria for the removal of outlier data and determination of mean yields and their variability. However, the standard only provides recommendations on outlier removal that should be taken into account by experts who undertake data interpretation. The potential for over-interpretation of data from small numbers of laboratories is highlighted and recommendations made to deal with this possibility.Key variables to the statistical analysis, the number of cigarettes per replicate and replicates performed in each laboratory, the number of participating laboratories and the use of linear and rotary smoking machines in smoke collection, are identified and their relevance to obtaining robust data are considered. The statistical methods routinely used for data analysis from the ISO regime, are re-assessed for their suitability to analyse data obtained under the Canadian intense (CI) regime, where yield differences between linear and rotary smoking machines are found. This machine effect can lead to more outliers being detected and difficulties in outlier detection which may affect the provision of robust estimates of mean yields, repeatability and reproducibility.