Health Canada Intense Research Articles

A Comparative In Vitro Toxicological Screening of a Closed-End Heated Tobacco Product *

SUMMARY Heated tobacco products (HTPs) are a recent category of tobacco products, with their relative safety compared to cigarette smoking and potential to help smokers to quit being two reasons why regulators may consider their market approval. Designed to heat tobacco rather than to burn in order to produce aerosol, different heating techniques are applied to commercial HTPs, which may result in differing aerosol formation. Therefore, each product requires separate assessment. This work focuses on a closed-end HTP (coded as HTP-A), which is electrically heated and designed to allow puffing air flow to bypass its tobacco section, resulting in reduced oxygen concentration within the tobacco section during heating and aerosol forming. To provide a preliminary aerosol chemistry and in vitro toxicological screening, this study assessed HTP-A against a commercial electrically heated HTP (IQOSTM, coded as HTP-B) and a 3R4F reference cigarette. Under Health Canada Intense (HCI) smoking regime, the levels of 9 regulatory priority toxicants in the aerosol of HTP-A were either reduced or comparable to those in HTP-B on a per-stick basis. Additionally, both HTPs showed significant reduction (greater than 90%) in comparison to those measured in mainstream smoke of 3R4F cigarette for these toxicants. Using a set of standard in vitro toxicological assays (Ames, Micronucleus and Neutral Red Uptake), the two HTPs showed no observable responses while significant toxicity responses were recorded for 3R4F’s total particulate matter. Based on these preliminary results, the novel closed-end HTP-A design may provide similar toxicological profiles to the comparator HTP-B. Further toxicological and clinical assessments are warranted to evaluate HTP-A’s potential for exposure or disease risk reduction. [Contrib. Tob. Nicotine Res. 32 (2023) 146–156]

Toxic mechanisms of cigarette smoke and heat-not-burn tobacco vapor inhalation on rheumatoid arthritis

Tobacco combustion exposure worsens rheumatoid arthritis (RA). Non-combustible tobacco devices, as heat-not-burn tobacco (HNBT), are emerging as harm reduction to smokers by releasing nicotine and lower combustible tobacco products. Nevertheless, HNBT toxicity remains unclear. Hence, here we investigated the impacts of the tobacco combustible product (cigarette smoke; CS) or HNBT vapor exposures on antigen-induced arthritis (AIA) in C57BL/6 mice. Animals were exposed to airflow, HNBT vapor, or CS during 1 h/twice a day, under the Health Canada Intense (HCI) smoking regime, between days 14 to 20 after the first immunization. At day 21, 16 h after the last exposures, mice were i.a. challenged and the AIA effects were evaluated 24 h later. CS- or HNBT-exposed mice presented equivalent blood nicotine levels. CS exposure worsened articular symptoms, pulmonary inflammation, and expression of lung metallothioneins. Nevertheless, CS or HNBT exposures reduced lymphoid organs' cellularity, splenocyte proliferation and IL-2 secretion. Additional in vitro CS or HNBT exposures confirmed the harmful effects on splenocytes, which were partially mediated by the activation of nicotine/α7nAchR pathway. Associated, data demonstrate the toxic mechanisms of CS or HNBT inhalation at HCI regime on RA, and highlight that further investigations are fundamental to assure the toxicity of emerging tobacco products on the immune system during specific challenges.

Robustness of HPHC Reduction in THS 2.2 Aerosol Relative to 3R4F Reference Cigarette Smoke under Extreme Climatic Conditions

Summary In order to assess robustness for the reduction of harmful and potentially harmful constituent (HPHC) levels generated by the Tobacco Heating System 2.2 (THS 2.2), a heated tobacco product, we compared the aerosol of this product with mainstream smoke from the 3R4F reference cigarette under different conditions of temperature and humidity. The desired climatic conditions were achieved by using an air-conditioning system coupled with the smoking-machine housing. Two extreme climatic conditions were selected, representing a “Hot and Dry” climate (30 °C and 35% relative humidity RH) and a “Hot and Very Humid” climate (30 °C and 75% RH). In addition, aerosol and smoke were generated using the standard conditions recognized for smoking-machine analyses of tobacco products (22 °C and 60% RH), which were close to the climatic conditions defined for “Subtropical and Mediterranean” environments (25 °C and 60% RH). The experimental conditions were chosen to simulate the use of THS 2.2 and cigarettes under extreme conditions of temperature and humidity. HeatSticks and cigarettes taken from freshly opened packs were subjected to short-term conditioning from two to a few more days under the same experimental conditions. We analyzed 54 HPHCs in THS 2.2 aerosol and 3R4F cigarette smoke, generated in accordance with the Health Canada Intense (HCI) standard, using modified temperature and humidity conditions for sample conditioning and machine-smoking experiments. We used a volume-adjusted approach for comparing HPHC reductions across the different climatic conditions investigated. Although a single puffing regimen was used, the total puff volume recorded for the 3R4F cigarette smoke varied due to the influence of temperature and humidity on combustion rate, which justified the use of a volume-adjusted approach. Volume-adjusted yields were derived from HPHC yields expressed in mass-per-tobacco stick normalized per total puff volume. The results indicated that, regardless of the considered climatic conditions, the HPHC levels investigated in THS 2.2 aerosol were reduced by at least 90%, on average, when compared with the concentrations in 3R4F cigarette mainstream smoke. This confirmed the robustness in performance for THS 2.2 to deliver reduced levels of HPHCs under the extreme climatic conditions investigated in this study. In order to further characterize the robustness of these reductions, the lowest reduction performance achieved for individual HPHCs across all climatic conditions was used to define the threshold for a robust reduction. The majority of the 54 HPHCs investigated in THS 2.2 aerosol showed more than 90% reduction. Calculations derived from nicotine-adjusted yields also confirmed robust reductions for all investigated HPHCs. The small differences in absolute reduction between the volume- and nicotine-adjusted approaches were predominantly attributed to a combination of the differences in both nominal nicotine deliveries and total puff volumes between THS 2.2 and 3R4F cigarettes; however, this did not influence the determination of robustness. Our findings confirm the value of this approach for assessing the robustness of a product’s performance under different climatic conditions.

In vitro toxicological evaluation of a tobacco heating product THP COO and 3R4F research reference cigarette on human lung cancer cells

Cigarette smoking increases health risks, such as respiratory diseases and heart diseases. Despite the decline in smoking rates in some countries, millions of adults still choose to smoke cigarettes. The use of next-generation nicotine delivery devices, such as tobacco heating products (THPs), may become a potentially safer alternative to smoking. Here, we report on the development of an electrically heated THP, coded as THP COO, with three different flavored tobacco sticks. The purpose of the study was to measure the levels of a list of harmful and potentially harmful constituents (HPHCs) in the total particulate matter (TPM) generated and to conduct a set of toxicological assessments of THP COO as compared with 3R4F reference cigarette. For all 55 HPHCs identified, the levels generated by the THP tobacco sticks were significantly lower in comparison to those in 3R4F TPM. The rate of reduction of HPHCs was between 68.6% and 99.9% under Health Canada Intense (HCI) smoking regimen. Human lung cancer cells (NCI-H292) exposed to 3R4F TPM showed dose-dependent responses for most of the 15 in vitro toxicity endpoints, whereas those exposed to comparable doses of THP COO TPMs did not. Therefore, exclusive use of the THP COO products may reduce the exposure of those tested HPHCs and thus potentially reduce health risk of smoking.

Differentiating between micronucleus dose-responses induced by whole cigarette smoke solutions with Benchmark Dose potency ranking

Dose-response modeling of in vitro micronucleus test (IVMNT) data was evaluated to determine if the approach has value in discriminating among different tobacco products. Micronucleus responses were generated in L5178Y/Tk+/− mouse lymphoma cells and TK6 human lymphoblastoid cells from a series of whole smoke solutions (WSSs) expected to have different levels of genotoxicity based on differences in their machine-generated smoke constituents. Eight WSSs were prepared by machine smoking different numbers (20 or 60) of two commercial cigarettes (Marlboro Silver or Red) under International Standardization Organization (ISO) or Health Canada Intense (HCI) smoking machine regimens and tested in the two cell lines with and without rat liver S9 activation. The S9-mediated IVMNT dose-response data from the WSSs were evaluated with PROAST software and Benchmark Doses (BMDs) and their upper and lower confidence intervals (CIs) were generated. IVMNT data differed based on the number and type of cigarettes smoked and smoking machine regimen. The IVMNT responses produced in mouse lymphoma cells generally were greater than in TK6 cells, but the ability of the two cell types to differentiate between WSSs was similar. The results indicate that BMD potency ranking was useful for differentiating between IVMNT responses.

Robustness of HPHC Reduction for THS 2.2 Aerosol Compared with 3R4F Reference Cigarette Smoke Under High Intensity Puffing Conditions

Summary In the absence of standards specific for testing the reduction robustness of the levels of harmful and potentially harmful constituents (HPHCs), the aerosol from the THS 2.2, a heated tobacco product, was compared with the mainstream smoke of the 3R4F reference cigarette over a broad range of machine-smoking regimes. The average reduction and the introduced concept of threshold limits of robust reduction were derived from HPHC concentrations, in mass per tobacco-stick normalized per total puff volume, to propose an alternative for the assessment of products where nicotine-adjusted yields would be inappropriate. In addition, this study explores the influence of 3R4F reference cigarette filter ventilation, and discusses the roles of temperature and precursors in the present context of robustness of HPHC reduction. Fifty-four HPHCs were analyzed under multiple regimes in THS 2.2 aerosol and 3R4F cigarette smoke. The average reduction of HPHC concentrations compared across all regimes characterized the robustness. Threshold limits of reduction of individual HPHCs were statistically determined across all regimes. The results observed under Health Canada Intense (HCI) and more intense regimes indicated that on average the reductions in HPHCs levels investigated in THS 2.2 aerosol were more than 90% and that the majority of the 54 HPHCs investigated in THS 2.2 aerosol showed more than 90% reduction. The robustness of THS 2.2 in maintaining the levels of reduction of representative HPHCs, whatever the puffing regime, can be quantified. The mass of HPHC per tobacco-stick normalized per total puff volume is a valuable approach to compare the robustness of the performance of a product over a large range of puffing conditions. Our findings will greatly complement the assessment for robustness of current and future similar products where classical approaches would present limitations.

Characteristic Human Individual Puffing Profiles Can Generate More TNCO than ISO and Health Canada Regimes on Smoking Machine When the Same Brand Is Smoked.

Human smoking behavior influences exposure to smoke toxicants and is important for risk assessment. In a prospective observational study, the smoking behavior of Marlboro smokers was measured for 36 h. Puff volume, duration, frequency, flow and inter-puff interval were recorded with the portable CReSSmicro™ device, as has often been done by other scientists. However, the use of the CReSSmicro™ device may lead to some registration pitfalls since the method of insertion of the cigarette may influence the data collection. Participants demonstrated consistent individual characteristic puffing behavior over the course of the day, enabling the creation of a personalized puffing profile. These puffing profiles were subsequently used as settings for smoking machine experiments and tar, nicotine and carbon monoxide (TNCO) emissions were generated. The application of human puffing profiles led to TNCO exposures more in the range of Health Canada Intense (HCI)-TNCO emissions than for those of the International Standardization Organization (ISO). Compared to the ISO regime, which applies a low puff volume relative to human smokers, the generation of TNCO may be at least two times higher than when human puffing profiles were applied on the smoking machine. Human smokers showed a higher puffing intensity than HCI and ISO because of higher puffing frequency, which resulted in more puffs per cigarette, than both HCI and ISO.

Integration of Time and Spatially Resolved In-Situ Temperature and Pressure Measurements With Soft Ionisation Mass Spectrometry Inside Burning Superslim and King-Size Cigarettes

Summary Background Combustion as well as pyrolysis of tobacco greatly affect the type and levels of toxicants in cigarette smoke. We previously developed an approach to combine simultaneous temperature and pressure measurements with fast in-situ microprobe chemical sampling inside a burning cigarette, producing a series of temperature and gas-flow velocity maps that characterize this dynamic system in response to externally applied air flow. Aim Two cigarette types differing only in diameter were puffed under ISO 3308 and Health Canada Intense (HCI) regimes to further understand the dynamic interaction of air flow and cigarette design parameters on tobacco combustion and pyrolysis by applying the thermophysical and thermo-chemical mapping approach. Methods Three types of sampling probes were inserted, which are thermocouple arrays for gas-phase temperature, quartz tubes for pressure measurement, and a heated sampling microprobe coupled to a single-photon soft ionisation mass spectrometer for chemical analysis. Two kinds of similarly constructed cigarettes with the same blend were analysed: superslim (17 mm circumference) and king-size (24 mm circumference). Synchronization among the sampled signals was achieved by mapping two probes (e.g., temperature/chemistry or temperature/pressure) at a time. The physical and chemical events were visualised and compared between the cigarettes and puffing regimes. Results A series of temperature, pressure, and chemical maps were obtained for the superslim and king-size cigarettes under ISO and HCI conditions. The pressure in the burning cigarette was higher in the superslim cigarette, and the temperature distribution differed between the two cigarette formats. As expected, temperatures and pressures were higher under HCI puffing than under ISO puffing for both cigarette formats. Thermochemical maps for e.g., benzene and nitric oxide formation were qualitatively similar between the superslim and king-size cigarettes. For other substances the distribution was markedly different. Conclusion The application of multi-probe in-situ chemical sampling is suitable to analyse highly dynamic combustion and pyrolysis processes occurring inside the two types of cigarettes. Ultimately, a direct comparison of cigarette circumferences on the complex combustion processes and formation of smoke constituents was achieved. [Beitr. Tabakforsch. Int. 29 (2020) 44–54]

An inter-laboratory in vitro assessment of cigarettes and next generation nicotine delivery products

In vitro testing can facilitate the rapid assessment of next generation nicotine delivery products (NGPs) with comparisons to combustible tobacco products. In vitro assays for cytotoxicity and oxidative stress were employed at BAT (UK) and JT (Japan) to test total particulate matter (TPM) of a scientific reference cigarette and aerosol collected mass (ACM) of a commercially available E-cigarette and two tobacco heating products (THP).3R4F TPMs were generated using the Health Canada intense (HCI) regimen, a modified regime (mHCI) for the THP ACMs and the CORESTA recommended method no. 81 for the E-cigarette ACM. Human lung cells were exposed to the test product TPM/ACMs at concentrations between 0–200 μg/ml followed by the employment of commercially available assays for endpoint analysis that included reactive oxygen species (ROS) generation, the glutathione ratio (GSH:GSSG), activation of the antioxidant response elements (ARE) and cellular viability. TPM/ACM nicotine concentrations were quantified using a UPLC-PDA technique.At both laboratories the 3R4F TPM induced significant and dose-dependent responses in all in vitro assays, whereas no significant responses could be measured for the NGP ACMs.In conclusion, both laboratories obtained comparable results across all endpoints therefore demonstrating the utility of the in vitro techniques combined with standardised test products to support the assessment of NGPs.

A Comparative Study of Non-Volatile Compounds Present in 3R4F Cigarettes and iQOS Heatsticks Utilizing GC-MS

It is now widely suggested that people who are dependent on nicotine should switch from ordinary tobacco smoking to alternative products, which at least reduce the overall harm from smoking. A number of alternatives are now popular, including electronic cigarettes and heatsticks. In this work comparative analysis of the smoke/aerosol emission from 3R4F standard cigarettes and iQOS heatsticks was undertaken. For this, gas chromatography-mass spectrometry (GC-MS) analysis was applied, to measure the non-volatile compounds of smoke/aerosol emission from individual samples, with the specific aim to determine their content of nicotine and selected other main components. All measurement data were collected under the Health Canada Intense (HCI) puffing regime. The most relevant findings of the present investigation can be summarized as follows. First, the number of measured aerosol components in the iQOS samples, with respect to those of 3R4F samples, was significantly lower (notably 37 versus 12 components). Second, the analysis of the iQOS and 3R4F GC-MS chromatographic fingerprints indicated a non-nicotine global component reduction (number and areas excluding nicotine) of larger than 80% for the iQOS samples in comparison to 3R4F samples. Third, the nicotine content of the iQOS aerosol was less than half that contained in the 3R4F smoke. The results from the present investigation indicate that—except for nicotine—smokers are exposed to a largely reduced number and amount of non-volatile, non-nicotine components in the iQOS heatstick aerosol, in comparison to those in the 3R4F cigarette smoke.

Little Cigars, Filtered Cigars, and their Carbonyl Delivery Relative to Cigarettes.

IntroductionLittle cigars and filtered cigars are currently growing in popularity due to their low cost and wide variety of flavors while retaining an appearance similar to cigarettes. Given the health consequences associated with cigarette use, it is important to understand the potential harm associated with these similar products. This includes the potential harm associated with carbonyls (eg, acetaldehyde, acrolein, formaldehyde, etc.), an important class of toxicants and carcinogens in tobacco smoke. Our objective was to determine the carbonyl levels in mainstream smoke from little and filtered cigars compared to cigarettes.MethodsWe examined two brands each of little cigars and filtered cigars, as well as two research cigarettes for carbonyl delivery using the International Organization of Standards (ISO) and the Health Canada Intense (HCI) machine-smoking protocols.ResultsOn a per puff basis, the levels of five of the seven carbonyls were higher from little cigars than filtered cigars and cigarettes (ISO: 56–116%; HCI: 39–85%; p < .05). On a per unit basis, most carbonyl levels were higher from both cigar types than cigarettes using the ISO method (ISO: 51–313%; p < .05) whereas only filtered cigars were higher using the HCI method (HCI: 53–99%; p < .05).ConclusionThese findings suggest that cigar smokers can be exposed to higher levels of carbonyls per cigar than cigarette smokers per cigarette.ImplicationsThese data will increase our understanding of the relative harm from carbonyl exposure from little and filtered cigars both for cigar-only smokers and the cumulative harm among the growing population of cigarette–cigar multi-product smokers.

Cigarette Filter Ventilation and Smoking Protocol Influence Aldehyde Smoke Yields.

The WHO study group on tobacco product regulation (TobReg) advised regulating and lowering toxicant levels in cigarette smoke. Aldehydes are one of the chemical classes on the TobReg smoke toxicants priority list. To provide insight in factors determining aldehyde yields, the levels of 12 aldehydes in mainstream cigarette smoke of 11 Dutch brands were quantified. Variations in smoking behavior and cigarette design affecting human exposure to aldehydes were studied by using four different machine testing protocols. Machine smoking was based on the International Standardization Organization (ISO) and Health Canada Intense (HCI) regime, both with and without taping the filter vents. The 11 cigarette brands differed in (i) design and blend characteristics; (ii) tar, nicotine, and carbon monoxide (TNCO) levels; (iii) popularity; and (iv) manufacturer. Cigarette smoke was trapped on a Cambridge filter pad and carboxen cartridge. After being dissolved in methanol/CS2 and derivatization with DNPH, the aldehyde yields were determined using HPLC-DAD. Using an intense smoking regime (increased puff volume, shorter puff interval) significantly increased aldehyde yields, following the pattern: ISO < ISO-taped < HCI-untaped < HCI. For all of the regimes, acetaldehyde and acrolein yields were strongly correlated (r = 0.804). The difference in TNCO and aldehyde levels between regular and highly ventilated low-TNCO cigarettes (as measured using ISO) diminished when smoking intensely; this effect is stronger when combined with taping filter vents. The highly ventilated low-TNCO brands showed six times more aldehyde production per mg nicotine for the intense smoking regimes. In conclusion, acetaldehyde and acrolein can be used as representatives for the class of volatile aldehydes for the different brands and smoking regimes. The aldehyde-to-nicotine ratio increased when highly ventilated cigarettes were smoked intensely, similar to real smokers. Thus, a smoker of highly ventilated low-TNCO cigarettes has an increased potential for higher aldehyde exposures compared to a smoker of regular cigarettes.

Selected Constituent Yield Variation in the Smoke of Commercial Cigarette Brands on the Japanese Market

Summary This study focused on the variation in the yields of constituents in smoke from commercial cigarette brands available on the Japanese market. Nineteen commercial cigarette brands were sampled five times every two months from 2009 to 2010. The target constituents were benzo[a]-pyrene, 1,3-butadiene, benzene, formaldehyde, acetaldehyde, acrolein, N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), carbon monoxide, “tar”, and nicotine. The results of this study showed that the coefficient of variation (CV) values varied greatly by brands, constituents, and smoking regimes. The yields of NNN and NNK in the smoke were strongly correlated to their yields in the tobacco filler blend for most brands. The yields of benzo[a]pyrene under the International Organization for Standardization (ISO) and the Health Canada Intense (HCI) smoking regimes and 1,3-butadiene under the HCI smoking regime were found to be influenced by the measurement. It was shown that factors for variation were highly varied among constituents. The grand mean of CV values for NNN and formaldehyde associated with cigarette manufacturing over ten months and measurement at the JT laboratory under the HCI smoking regimes were 17.1% and 6.6% respectively. The grand mean of CV values for NNN and formaldehyde associated with both cigarette manufacturing over ten months and measurement at different laboratories under the HCI smoking regimes were 23.7% and 22.9% respectively. This is due to the fact that formaldehyde showed the highest CV values for reproducibility among the constituents. Thus, in order to set realistic and robust confidence intervals, it is very important to take into account the variations associated with cigarette manufacturing and measurement within and between laboratories.

Effects of Topography-Related Puff Parameters on Carbonyl Delivery in Mainstream Cigarette Smoke.

Smoking topography parameters differ substantially between individual smokers and may lead to significant variation in tobacco smoke exposure and risk for tobacco-caused diseases. However, to date, little is known regarding the impact of individual puff parameters on the delivery of many harmful smoke constituents including carbonyls. To examine this, we determined the effect of altering individual puff parameters on mainstream smoke carbonyl levels in machine-smoked reference cigarettes. Carbonyls including formaldehyde, acetaldehyde, crotonaldehyde, propionaldehyde, methyl ethyl ketone (MEK), acrolein, and acetone were determined in cigarette smoke by HPLC after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Deliveries of all carbonyls were nearly two-fold greater when cigarettes were smoked according to the more intense Health Canada Intense (HCI) protocol compared to the International Organization of Standardization (ISO) method, consistent with the two-fold difference in total puff volume between methods (ISO: 280-315 mL; CI: 495-605 mL). When individual topography parameters were assessed, changes in puff volume alone had the greatest effect on carbonyl delivery as predicted with total carbonyls being strongly correlated with overall puff volume (r2: 0.52-0.99) regardless of how the differences in volume were achieved. All seven of the carbonyls examined showed a similar relationship with puff volume. Minor effects on carbonyl levels were observed from vent blocking and changing the interpuff interval, while effects of changing puff duration and peak flow rate were minimal. Overall, these results highlight the importance of considering topography, especially puff volume, when the toxicant delivery and potential exposure smokers receive are assessed. The lack of an impact of other behaviors, including puff intensity and duration independent of volume, indicate that factors such as temperature and peak flow rate may have minimal overall effects on carbonyl production and delivery.

A comparative assessment of e-cigarette aerosols and cigarette smoke on in vitro endothelial cell migration

Cigarette smoking is a risk factor for several diseases. There has been a steep increase in the use of e-cigarettes that may offer a safer alternative to cigarette smoking. In vitro models of smoking-related diseases may provide valuable insights into disease mechanisms associated with tobacco use and could be used to assess e-cigarettes.We previously reported the application of a ‘scratch wound’ assay, measuring endothelial cell migration rate following artificial wounding, in the presence or absence of cigarette smoke extracts. This study reports the comparative effects of two commercial e-cigarette products (Vype ePen and Vype eStick) and a scientific reference cigarette (3R4F) on endothelial migration in vitro.Puff-matched extracts were generated using the Health Canada Intense (HCI) regime for cigarettes and a modified HCI for e-cigarettes. Exposure to 3R4F extract (20h) induced concentration-dependent inhibition of endothelial cell migration, with complete inhibition at concentrations >20%. E-cigarette extracts did not inhibit migration, even at double the 3R4F extract nicotine concentration, allowing cells to migrate into the wounded area.Our data demonstrate that e-cigarettes do not induce the inhibition of endothelial cell migration in vitro when compared to 3R4F. The scratch wound assay enables the comparative assessment between tobacco and nicotine products in vitro.

Characterization and Application of the VITROCELL VC1 Smoke Exposure System and 3D EpiAirway Models for Toxicological and e-Cigarette Evaluations

Abstract The VITROCELL VC1® smoke exposure machine is an aerosol exposure system that has been used to conduct toxicological assessments with cigarettes and e-cigarettes and can be used with the human 3D EpiAirway™ tissue model (MatTek Corporation). The goals of this study were to assess exposure parameters of the VC1, and evaluate donor-to-donor variability of three EpiAirway tissue donors with the following endpoints: viability, barrier integrity, and gene promoter/expression regulation. Additionally, we applied the EpiAirway model to assess the effects of aerosol from two reference combustible cigarettes and two e-cigarettes. Whole smoke (WS) exposures with Kentucky Reference 3R4F cigarettes and Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Monitor were conducted under International Organization for Standardization or Health Canada Intense (HCI) conditions, and e-cigarette exposures were conducted under modified HCI conditions. Particulate deposition and collection were consi...

Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol

The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under “desert” or “tropical” conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine-smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen.

A modified trapping method to quantify hydrogen cyanide in mainstream and sidestream cigarette smoke under ISO and Health Canada intense smoking regimes

Hydrogen cyanide is a toxic compound and plays a critical role in cigarette smoke hazard assessment. Due to its reactivity with other chemicals of cigarette smoke, hydrogen cyanide collection was accomplished by the novel trapping method established in our laboratory using glass-fibre filter pads (GFP) treated by sodium hydroxide. However, the GFP trapping method has not been tested under Health Canada Intense (HCI) smoking regime and the trapping efficiency of GFP versus traditional methods have not been evaluated. The present study employed the two trapping methods to collect hydrogen cyanide in mainstream and sidestream cigarette smoke under ISO and HCI smoking regimes. The causes leading to losses of hydrogen cyanide and effects of smoking parameters used in the traditional trapping method were also investigated in this study. It was found that under both ISO and HCI smoking regimes the amounts of hydrogen cyanide in GFP trapping method were strongly correlated (r>0.99) with those by the traditional trapping method, though the traditional method trapped less hydrogen cyanide in sidestream smoke. Carbonyl compounds, such as formaldehyde, were identified as the contributors for the loss of hydrogen cyanide in the alkaline solution. Puff profile was affected to some extents by the use of impinger. Collectively, the results indicate that the modified trapping method is preferred for the routine analysis of hydrogen cyanide in cigarette smoke under ISO and HCI smoking regimes.

Tobacco-specific N-nitrosamines NNN and NNK levels in cigarette brands between 2000 and 2014

The evolution of the levels of tobacco-specific N-nitrosamines (TSNA), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mainstream (MS) cigarette smoke is investigated based on smoke and tobacco chemistry data of cigarette brands sold by Philip Morris International (PMI) between 2000 and 2014. A total of 315 cigarette samples representing a wide range of product and design characteristics manufactured by PMI between 2008 and 2014 were analyzed and compared to a previously published dataset of PMI brands manufactured in 2000. The data indicate that there is a substantial reduction of NNN and NNK levels in tobacco fillers and MS cigarette smoke per mg of tar and per mg of nicotine using Health Canada Intense (HCI) machine-smoking regime. This observed reduction in NNN and NNK levels in MS cigarette smoke is also supported by the downward trend observed on NNN and NNK levels in USA flue-cured Virginia and Burley tobacco lots from 2000 to 2014 crops, reflecting effectiveness of measures taken on curing and agricultural practices designed to minimize TSNA formation in tobacco.

Five-year yield variation in N-nitrosonornicotine and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone from the smoke of commercial cigarette brands on the Japanese market

This study reports on variation in the yields of N-nitrosonornicotine (NNN) and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) from the mainstream smoke of cigarette brands and describes factors affecting this variation. Yields of NNN and NNK from smoke and tobacco filler, which is blended and cut tobacco filled in a cigarette tube, together with cigarette design parameters, were measured for 11 commercial cigarette brands, which were available in Japan from 2009 to 2013. The ranges of the relative standard deviations (RSDs) for NNN from the smoke of each brand were 5.0%–29.9% under International Organization for Standardization (ISO) and 6.3%–26.3% under Health Canada Intense (HCI) smoking regime. When these RSDs were compared with those of tar, nicotine, and carbon monoxide yields in each brand, they were found to be much higher for all of the brands under the HCI, and higher for most of the brands under the ISO smoking regime. In addition, the RSDs of NNN and NNK in smoke were mostly higher than those of KY3R4F which was manufactured in a single batch. It was identified that variation in NNN yields from tobacco filler mainly contribute to variation in NNN yields from smoke.