Health Canada Intense Research Articles

Empirical characterisation of ranges of mainstream smoke toxicant yields from contemporary cigarette products using quantile regression methodology

Approximately 100 toxicants have been identified in cigarette smoke, to which exposure has been linked to a range of serious diseases in smokers. Smoking machines have been used to quantify toxicant emissions from cigarettes for regulatory reporting. The World Health Organization Study Group on Tobacco Product Regulation has proposed a regulatory scenario to identify median values for toxicants found in commercially available products, which could be used to set mandated limits on smoke emissions. We present an alternative approach, which used quantile regression to estimate reference percentiles to help contextualise the toxicant yields of commercially available products with respect to a reference analyte, such as tar or nicotine. To illustrate this approach we examined four toxicants (acetone, N′-nitrosoanatabine, phenol and pyridine) with respect to tar, and explored International Organization for Standardization (ISO) and Health Canada Intense (HCI) regimes. We compared this approach with other methods for assessing toxicants in cigarette smoke, such as ratios to nicotine or tar, and linear regression. We concluded that the quantile regression approach effectively represented data distributions across toxicants for both ISO and HCI regimes. This method provides robust, transparent and intuitive percentile estimates in relation to any desired reference value within the data space.

Transient measurement of the effective particle density of cigarette smoke

The real-time effective particle density of cigarette smoke was determined using a Centrifugal Particle Mass Analyzer (CPMA) and Differential Mobility Spectrometer (DMS). A Puff Inhale Exhale (PIE) simulator was used to produce the smoke from various research and commercial cigarettes following the International Standard Organization (ISO) puffing parameters (35ml puff of 2s duration, every 60s) or the Health Canada Intense (HCI) puffing parameters (55ml puff of 2s duration, every 30s). The impact of modifying parameters, such as smoke mass, cigarette format, filter type, inhalation volume and mouth hold period, on the effective particle density was also investigated. All of the effective density functions were found to be independent of particle size within the bias uncertainty of the measurement system, indicating that the cigarette smoke particles likely had a spherical morphology. Trends in the average effective particle densities were observed for the different cigarettes and puffing parameters. While all of these shifts were within the bias uncertainty of the CPMA–DMS system, two-sample t-tests and the Tukey method were used to identify where the shifts were statistically probable. However due to the complexity of cigarette smoke, the aerosol mechanisms behind most of these shifts were unknown and require further investigation. For all of the tested cases the average effective particle density, considering puffs 3–6, varied from 1090 to 1518kg/m3, with a majority (9 out of 16 cases) falling within 1300 to 1394kg/m3. The Tukey method identified no statistical change in the effective particle density over the duration of an ISO puff, but it did identify significant differences between effective densities produced by different cigarettes.

Hygroscopic effects on the mobility and mass of cigarette smoke particles

The hygroscopic growth of particles, produced from a University of Kentucky 3R4F reference cigarette smoked following Health Canada Intense (HCI) puffing parameters (55mL puff of 2s duration, every 30s), was measured in terms of the electrical mobility diameter and particle mass, using a Hygroscopic Tandem Differential Mobility Analyzer (HTDMA) and Centrifugal Particle Mass Analyzer (CPMA) system. Both the particle mobility diameter and mass growth factors were found to agree with previously determined values and hygroscopicity models. The mobility diameter growth factor of the particles produced from either a University of Kentucky 3R4F or 1R5F reference cigarette, following HCI puffing parameters, were found to be very similar. As the relative humidity (RH) approached saturation, the effects of the initial particle size on the mobility growth factor became more dominant, with larger particles growing proportionally larger than smaller particles. From the measured mobility diameter and mass growth factors, the density growth factor was calculated. This parameter showed that the particle density increased as the sample relative humidity increased. This case is only possible, given that the dried smoke particle density (1109±118kg/m3) was determined to be greater than the density of water, if the water condensation on the smoke particle dissolves at least a portion of it, resulting in a significant increase in mass with only a small increase volume.

An Improved Theoretical Model of Cigarette Smoke Filtration across Mono-Segment Cellulose Acetate Filters

Summary An improved theoretical model was presented to predict the filtration efficiency of cigarette filters. Filtration equations of single fibers considering the interference of neighboring fibers were applied in the model. Cellulose acetate fibers in cigarette filters were approximated as cylinders. The fiber size was adjusted by its size projected on the flow field. The solid fraction of fibers in cigarette filters was recalculated using the size of the virtual cylinders. The varying flow velocity during smoking was taken into account when calculating the filtration efficiency. The effective hydrodynamic particle diameter of cigarette smoke was estimated to be 0.44 μm by the difference of filtration efficiencies under ISO and Health Canada Intense (HCI) smoking regimes. Filtration contributions due to diffusion, interception and inertial impaction were 62%, 32% and 6%, respectively, at a flow velocity of 0.38 m/s for particles of 0.44 μm diameter. The effect of inertial impaction was insignificant but not negligible under ISO smoking regime. The measured and predicted efficiencies of two cigarette samples were compared and satisfactory agreement was obtained. [Beitr. Tabakforsch. Int. 26 (2015) 232-240]

Measurement of Chlorinated Dioxins and Furans in Cigarette Mainstream Smoke

Summary Chlorinated dioxins and furans (‘dioxins’) are not ordinarily detected in cigarette smoke at levels likely to exceed the recommended ‘safe’ limits recognised by health and environmental protection agencies. By convention they are measured in terms of their total toxic equivalence (TEQ). The method developed by this group utilises high sensitivity gas chromatography-tandem mass spectrometry (GCMS/ MS). The limit of detection (LOD) was determined to be 4.3 pg TEQ/cig; under ISO 3308 smoking conditions. Dioxins are not subject to any routine regulatory control in cigarette smoke. However, certain dioxin congeners are highly toxic and they have been cited by the United States Food and Drug Administration (FDA) as harmful and potentially harmful constituents (HPHC) of tobacco smoke. This raises the issue of how to monitor and measure uncertainty when the analyte(s) of interest are not normally present in the matrix of interest. In the absence of a reference cigarette containing levels of dioxins above the detection limit; we prepared test pieces containing known quantities of 17 dioxin congeners, regarded by the World Health Organisation (WHO) as significantly toxic. This paper provides data for the transfer rates performed under ISO 3308 (ISO) and Health Canada Intense (HCI) machinesmoking conditions; and proposes a means to estimate the uncertainty (U) based on TEQ. [Beitr. Tabakforsch. Int. 26 (2015) 214-218]

Differences in cadmium transfer from tobacco to cigarette smoke, compared to arsenic or lead

Arsenic, cadmium and lead levels in tobacco filler and cigarette smoke were determined in a 568-sample worldwide survey. Median tobacco levels for arsenic, cadmium and lead were 237, 769 and 397ng/g respectively, comparable to those previously reported albeit somewhat lower for lead and cadmium. Median mainstream smoke yields for arsenic, cadmium and lead were <3.75, 18.2, and <12.8ng/cig. under ISO, and <8.71, 75.1 and <45.7ng/cig. under Health Canada Intense (HCI) smoking regime respectively. In the case of cigarettes with activated carbon, a selective retention of cadmium but not lead or arsenic was observed. This effect was more pronounced under ISO than under HCI smoking regimes. Cadmium selective retention by activated carbon was confirmed by testing specially designed prototype cigarettes and the causes for this selective filtration were investigated. The differences between cadmium, arsenic and lead in terms of their speciation in tobaccos and in cigarette smoke could be related to their distribution in the ash, butt, mainstream (in gas-phase and particulate-phase) and sidestream smoke of a smoked cigarette. The possible formation of organometallic cadmium derivatives in the smoke gas-phase is discussed, the presence of which could adequately explain the observed cadmium selective filtration.

Analysis of Vinyl Chloride in Vapor Phase of Mainstream Cigarette Smoke by GC–MS Under ISO and Health Canada Intensive Machine Smoking Regimens

Vinyl chloride (VC) in the vapor phase of mainstream cigarette smoke was determined under both International Organization for Standardization (ISO) and Health Canada intensive (HCI) machine smoking regimens, which was suspected to be carcinogenic compound. VC was collected by passing the mainstream cigarette smoke through a Cambridge Filter Pad (CFP) into cryogenic traps containing methanol. The impinger solutions were fortified with VC-d 3 and analyzed by GC–MS. Limits of detection for vinyl chloride was 0.9 ng mL−1 with the recovery in the range of 93.2–98.4 %. Moreover, the intra-day and inter-day precision was 7.39 and 9.77 %, respectively. Under HCI machine smoking regimen, the vinyl chloride yields in vapor phase of mainstream cigarette smoke were much higher and the average increase was greater than 100 % compared with those under ISO smoking condition.

Comparison of dermal tumor promotion activity of cigarette smoke condensate from prototype (heated) cigarette and reference (combusted) cigarette in SENCAR mice

Test cigarette (prototype “heated” cigarette) was evaluated on its dermal tumor promotion activity in SENCAR mice relative to conventional 3R4F cigarette. Mainstream cigarette smoke was generated under the modified Health Canada Intensive Regimen, and smoke condensate (CSCs) were collected using cold traps and extracted with acetone. Female mice received a topical application of 7,12-dimehtylbenz(a)anthracene (DMBA) as the tumor initiator on the back skin during Week 1. Subsequently, CSC was repeatedly applied as the tumor promoter at 5 doses, up to 30mg tar/application, three times per week for 30weeks. Test groups showed a clearly longer latency at lower doses (⩽15mg), but the difference was less clear at higher doses (⩾22.5mg), while mortalities were not affected throughout the study. Test groups also had consistently lower incidence and multiplicity of neoplasms, as well as lower incidences of non-neoplastic changes (e.g., inflammations and squamous epithelial hyperplasia on the site of application). The group without DMBA initiation did not induce any neoplasm but the respective Reference group showed an increase in tumorigenicity. In conclusion, the study demonstrated significant reduction in dermal irritancy and tumorigenicity of Test CSC compared to Reference CSC.

Mutagenicity of smoke condensates from Canadian cigarettes with different design features

There is currently limited knowledge regarding the impact of different cigarette designs on the toxicological properties of cigarette smoke condensate (CSC). This study used the Salmonella Mutagenicity Assay to examine the mutagenic activity of mainstream CSCs from 11 commercial Canadian cigarette brands with different design features or tobacco blend. The brands were selected to include design features that are common for cigarettes sold in the Canadian market, as well as cigarettes with alternate filters (charcoal or MicroBlue™), the super slim design, and cigarettes containing mixed blends of different tobacco types. CSCs were obtained using the International Organization for Standardization (ISO) and Health Canada Intense (HCI) smoking regimes, and mutagenic activity was assessed using Salmonella strains TA98, YG1041 and YG5185. Comparisons of the commercial brands to the Kentucky 3R4F, the Canadian Monitor 8 reference and a Canadian best seller revealed no significant reduction in CSC mutagenicity for cigarettes with alternate filters. However, the super slim design did afford some reduction in mutagenic potency. Nevertheless, since the study did not attempt to evaluate the impact of the cigarette designs on human health at the individual or population level, the super slim cigarettes cannot be considered 'reduced-harm' cigarettes.

Quantitative Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) Cited by the United States Food and Drug Administration

Abstract The yields of 16 polycyclic aromatic hydrocarbons (PAHs) were determined from cigarette mainstream smoke condensate extracts using Gas Chromatography- Tandem Mass Spectrometry (GC-MS/MS). The method has been validated for ISO and Health Canada Intense (HCI) smoking protocols. Quantifiable levels (ISO means 0.16 to 365 ng/cig; HCI means 0.33 to 1595 ng/cig; n = 30) of 15 PAHs were found in the Kentucky reference cigarette K3R4F. The coefficient of variance (CV) was derived from ten determinations each run in triplicate. The CV range was 8.7% to 24.8% (ISO) and 6.6% to 24.3% (HCI). The limit of detection (LOD) based on empirical precision was ≤ 0.06 ng/cig (ISO) and ≤ 0.20 ng/cig (HCI) for all components except naphthalene (2.89 and 9.62 ng/cig, respectively). The yields from 5 unspecified branded cigarettes (Samples A-E) and 2 other reference cigarettes, K1R5F and the CORESTA monitor CM7, were determined under ISO smoking conditions. The same 15 PAHs were detected as in the K3R4F; however, cigarettes with lower yields of total particulate matter (TPM) were found to contain significantly less PAHs. One component was measured below the limit of quantification (LOQ) in Sample E and 2 components were &lt; LOQ in the K1R5F.

Smoking topography and biomarkers of exposure among Japanese smokers: associations with cigarette emissions obtained using machine smoking protocols

Although the relative risk of lung cancer due to smoking is reported to be lower in Japan than in other countries, few studies have examined the characteristics of Japanese cigarettes or potential differences in smoking patterns among Japanese smokers. To examine tar, nicotine and carbon monoxide (TNCO) emissions from ten leading cigarettes in Japan, machine smoking tests were conducted using the International Organization for Standardization (ISO) protocol and the Health Canada Intense (HCI) protocol. Smoking topography and tobacco-related biomarkers were collected from 101 Japanese smokers to examine measures of exposure. The findings indicate considerable variability in the smoking behavior of Japanese smokers. On average, puffing behaviors observed among smokers were more similar to the parameters of the HCI protocol, and brands with greater ventilation that yielded lower machine values using the ISO protocol were smoked more intensely than brands with lower levels of ventilation. The smokers of "ultra-low/low" nicotine-yield cigarettes smoked 2.7-fold more intensively than those of "medium/high" nicotine-yield cigarette smokers to achieve the same level of salivary cotinine (p=0.024). CO levels in expiratory breath samples were associated with puff volume and self-reported smoking intensity, but not with nominal values of nicotine-yield reported on cigarette packages. Japanese smokers engaged in "compensatory smoking" to achieve their desired nicotine intake, and levels of exposure were greater than those suggested by the nominal value of nicotine and tar yields reported on cigarette packages.

Mainstream Smoke Chemistry and in Vitro and In Vivo Toxicity of the Reference Cigarettes 3R4F and 2R4F

Abstract A new reference cigarette, the 3R4F, has been developed to replace the depleting supply of the 2R4F cigarette. The present study was designed to compare mainstream smoke chemistry and toxicity of the two reference cigarettes under the International Organization for Standardization (ISO) machine smoking conditions, and to further compare mainstream smoke chemistry and toxicological activity of the 3R4F cigarette by two different smoking regimens, i.e., the machine smoking conditions specified by ISO and the Health Canada intensive (HCI) smoking conditions. The in vitro cytotoxicity and mutagenicity was determined in the neutral red uptake assay, the Salmonella reverse mutation assay, and the mouse lymphoma thymidine kinase assay. Additionally, a 90-day nose-only inhalation study in rats was conducted to assess the in vivo toxicity. The comparison of smoke chemistry between the two reference cigarettes found practically the same yields of total particulate matter (TPM), ‘tar’, nicotine, carbon monoxide, and most other smoke constituents. For both cigarettes, the in vitro cytotoxicity, mutagenicity, and in vivo toxicity showed the expected smoke-related effects compared to controls without smoke exposure. There were no meaningful differences between the 2R4F and 3R4F regarding these toxicological endpoints. The assessments for the 3R4F cigarette by smoking regimen found as a trivial effect, due to the higher amount of smoke generated per cigarette under HCI conditions, an increased yield of toxicant and higher toxicological activity per cigarette. However, per mg TPM, ‘tar’, or nicotine, the amounts of toxicants and the in vitro toxicity were generally lower under HCI conditions, but the in vivo activity was not different between the two machine smoking conditions. Overall, as the main result, the present study suggests equivalent smoke chemistry and in vitro and in vivo toxicity for the 2R4F and 3R4F reference cigarettes.

Effects of smoking regimens and test material format on the cytotoxicity of mainstream cigarette smoke

The purpose of this study was to evaluate the effects of test material format and smoking regimens on comparative toxicity testing of cigarette smoke. Total particulate matter (TPM) or whole smoke (WS) generated from three test cigarettes under International Organization for Standardization (ISO) or Health Canada Intensive (HCI) regimens were assessed for cytotoxicity using the neutral red uptake (NRU) cytotoxicity assay. Under both ISO and HCI regimens, the relative differences of cytotoxicity among the test cigarettes indicated by the EC50 values in WS were significantly higher than those in TPM. For TPM testing, cytotoxicity was decreased going from ISO regimen to HCI regimen, consistent with the reported reductions of toxicant output on a per unit of TPM basis under the HCI regimen. For WS, cytotoxicity increased for the two lower TPM cigarettes, and decreased for the higher TPM cigarette going from HCI regimen to ISO regimen. Results from this study demonstrated WS should be the preferable test material format for smoke toxicity testing whenever possible. Intensive smoking regimens, such as HCI, are less likely to underestimate smoke toxicant intakes by smokers, and should be included in the comparative toxicological testing strategy.

Design and chemical evaluation of reduced machine-yield cigarettes

Experimental cigarettes (ECs) were made by combining technological applications that individually reduce the machine measured yields of specific toxicants or groups of toxicants in mainstream smoke (MS). Two tobacco blends, featuring a tobacco substitute sheet or a tobacco blend treatment, were combined with filters containing an amine functionalised resin (CR20L) and/or a polymer-derived, high activity carbon adsorbent to generate three ECs with the potential for generating lower smoke toxicant yields than conventional cigarettes. MS yields of smoke constituents were determined under 4 different smoking machine conditions. Health Canada Intense (HCI) machine smoking conditions gave the highest MS yields for nicotine-free dry particulate matter and for most smoke constituents measured. Toxicant yields from the ECs were compared with those from two commercial comparator cigarettes, three scientific control cigarettes measured contemporaneously and with published data on 120 commercial cigarettes. The ECs were found to generate some of the lowest machine yields of toxicants from cigarettes for which published HCI smoke chemistry data are available; these comparisons therefore confirm that ECs with reduced MS machine toxicant yields compared to commercial cigarettes can be produced. The results encourage further work examining human exposure to toxicants from these cigarettes, including human biomarker studies.

Mutagenic, cytotoxic, and genotoxic properties of tobacco smoke produced by cigarillos available on the Canadian market

Cigarillos (aka little cigars) have been increasing in popularity unlike cigarettes; but relatively little is known about the toxicology of the mainstream smoke (MSS) from such products. Therefore, the objective of this work was to compare the toxicological properties of the MSS (Health Canada Intensive smoking conditions) from a range of cigarillo products with the toxicological properties of MSS of cigarettes. Three in vitro assays were used to evaluate the toxicities of the MSS total particulate matter (TPM): (1) mutagenicity using Ames assay with Salmonella strains TA98 and TA100 with S9 metabolic activation (+S9); (2) cytotoxicity using the Neutral Red Uptake (NRU) assay with CHO (Chinese Hamster Ovary) cells; and (3) genotoxicity using the micronucleus assay with CHO cells and short-term exposures (3-h±S9). The Ames assay (TA100+S9) and the NRU assay were also applied to the gas/vapour phase of the MSS that passed through the Cambridge pad. On a per-milligram-nicotine basis, the preferred way of comparing toxicities of different types of tobacco products, the MSS from cigarillos was not less toxic, and in some cases more toxic (TPM fraction TA98+S9, NRU), than the MSS from cigarettes. Thus, our findings support our prior work on smoke mutagenicity that showed MSS from cigarillos was not less toxic than MSS from cigarettes.

Free-base nicotine in tobacco products. Part I. Determination of free-base nicotine in the particulate phase of mainstream cigarette smoke and the relevance of these findings to product design parameters

The free-base nicotine (FBN) content of mainstream cigarette smoke (MSS) has been discussed in the peer-reviewed literature and popular press. It has been alleged that manufacturers adjust product design features to increase the percentage of total nicotine (TN) in the MSS gas–vapor phase that is unprotonated [ P g ,nic(%)] and/or the fraction of nicotine in the MSS total particulate matter (TPM) that is unprotonated (FBN/TN). Our research showed the Health Canada Intensive smoking conditions negated the effects of blend and cigarette design features reported to raise the pH of TPM collected under ISO or US FTC conditions. Our research also showed that when additive-free Canadian cigarettes were smoked under ISO conditions, the FBN/TN ratio increased as the tar/nicotine ratio decreased. Our findings are in line with other studies that have questioned allegations of a relationship between use of ammonia and its compounds as tobacco additives and amounts of unprotonated nicotine in MSS. In addition, the experimental work demonstrated how use of solid-phase microextraction to estimate FBN can yield erroneously high results due to improper conditioning and/or smoking of the cigarettes. Our research showed that there is no longer any scientific support for regulators to require smoke pH and FBN determinations on cigarette products.

In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens

Previous studies on the biological assessment of cigarette smoke (CS) mainly focused on the total particulate matter (TPM) collected using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS). To study the effects of native CS in vitro, direct exposure methods have been developed. Meanwhile, in vitro micronucleus (MN) assays have been reported to evaluate the mutagenicity of CS. The objective of this research is to investigate the MN-inducing activity of whole smoke (WS) and GVP using a whole smoke exposure system, CULTEX ®, which allows direct exposure of cultured cells to native CS at the air/liquid interface (ALI). CS was generated according to the International Organization for Standardization (ISO; 35 ml, 2 s, once per 60 s) or the Health Canada Intensive (HCI; 55 ml, 2 s, once per 30 s, with complete ventilation block) regimens and Chinese hamster lung (CHL/IU) cells were then exposed to this smoke. Dosages were adjusted according to the amount of smoke entering the actual exposure position. Under both smoking regimens, WS and GVP from 2R4F reference cigarettes induced MN responses. The concept of the dosage and similar dose–response relationships between theoretical and monitored dosage values under the two regimens enabled us to compare the MN-inducing activities of cigarettes in the direct exposure assay, even in the case of various experimental settings or different TPM amounts. MN-inducing activities of 2R4F under the ISO regimen seemed to be higher than those under HCI estimated by the TPM equivalent calculated values.

Nicotine, Tar, and Mutagenicity of Mainstream Smoke Generated by Machine Smoking with International Organization for Standardization and Health Canada Intense Regimens of Major Japanese Cigarette Brands

Based upon the Framework Convention on Tobacco Control (FCTC), the World Health Organization (WHO) ha sr ecommended that health authorities disclose toxicological properties of cigarette mainstream smoke (MSS) obtained not only according to US Federal Trade Commission (FTC)/International Organization for Standardization (ISO) conditions but also by more intense conditions such as the Health Canada Intense (HCI) condition. This is because smokers are believed to smoke more intensely than machine smoking under the ISO regimen. Because there are no previous reports on the toxicological properties of MSS of Japanese cigarettes under the HCI condition, we determined nicotine and water contents by gas chromatography (GC-MS and GC/Thermal Conductivity Detector (TCD)) for three product lots each of the ten bestselling brands of Japanese cigarettes following the WHO protocol. One of the three lots of each MSS condensate was also resolved in dimethylsulfoxide and investigated by Ames preincubation assay using Salmonella typhimurium TA100, TA98, and YG1024 strains with and without metabolic activation (rat liver S9 mix). Nicotine and tar yields with the HCI regimen were higher than those with ISO, the latter being very close to the values described on the packages of each cigarette brand. Mutagenicity was mainly observed in TA98 and YG1024 with metabolic activation. Mutagenic activity of MSS with the HCI regimen was 1.4–9 times higher than that with the ISO regimen. Based on YG1024 with activation, high activities were observed in several “low yield” brands. The activity of “low-yield” brands with the HCI regimen was not always lower than that of regular-yield brands with the ISO regimen. These results suggest that “low yield” cigarettes do not result in reduced exposure or reduced risk to humans.

Reductions in the tobacco specific nitrosamine (TSNA) content of tobaccos taken from commercial Canadian cigarettes and corresponding reductions in TSNA deliveries in mainstream smoke from such cigarettes

Tobacco specific nitrosamines (TSNAs) are suspected to cause smoking-related neoplastic diseases. The change from direct-fired to indirect-fired barns (aka kilns) for curing bright (aka Virginia, flue-cured) tobaccos was made to reduce the TSNA concentrations. The effectiveness of such processes in reducing the deliveries of TSNAs to the users of the products should be monitored. However, it is difficult to assess the effects of this reduction on the TSNA levels in mainstream smoke when cigarette blends contain burley tobaccos and other blend components that can increase smoke TSNA concentrations. Canadian cigarettes made prior to and in the few years just after the conversion to indirect-fired curing should not be subject to such interferences. Thus, the TSNA content of tobaccos and mainstream smoke from six brands of Canadian cigarettes produced in 2003, 2004, and 2005 were determined. Reductions in NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], the most important TSNA in flue-cured tobaccos, levels in the tobacco blends ranged from 60% to 85%. The corresponding reductions in mainstream smoke TSNA levels ranged from 59% to 72% (ISO smoking conditions) and 58-76% (Health Canada Intensive smoking conditions). These results show that other factors (microorganisms, nitrite levels) may be negating the TSNA reductions achieved by indirect-fired curing.

Effect of Smoking Conditions and Methods of Collection on the Mutagenicity and Cytotoxicity of Cigarette Mainstream Smoke

There is a history for the use of in vitro bioassays to assess the toxicological properties of mainstream cigarette smoke (MSS). The results described in the literature were, for the most part, obtained with MSS collected under Federal Trade Commission (FTC) or International Organization for Standardization (ISO) conditions. However, numerous studies have shown that smokers smoke their cigarettes more intensely (e.g., they take larger puffs and/or more frequent puffs and/or partially occlude filter ventilation) than they are smoked on smoking machines operated under FTC (or ISO) conditions. It has also been reported that MSS composition changes with changes in smoking conditions. Furthermore, some governmental agencies have adopted regulations that specify more intensive protocols (i.e., Health Canada Intensive, HCI) for the collection of MSS for in vitro toxicological assays. Consequently, the performance of the Ames assay (TA98+S9, TA100+S9) and neutral red uptake assay under ISO and HCI protocols was studied with two blended (KR1R4F/KR2R4F, KR1R5F) and one flue-cured (CIM-7) reference cigarettes. The main outcome was when results were reported on a per milligram TPM (that portion of the mainstream smoke which is trapped in the smoke trap, expressed as milligrams per cigarette) basis generated under ISO conditions was more mutagenic and more cytotoxic than was TPM generated under HCI conditions. However, the decrease in biological activity could not be explained only by the increased in the water content of the TPM on going from ISO to HCI smoking conditions, and the results may be influenced by differences in smoke chemistry as a result of differing smoke collection systems.