Test Cigarettes Research Articles

Direct introduction of cigarette smoke for puff-by-puff trace metals analysis by inductively coupled plasma mass spectrometry

Puff-by-puff on-line analysis of trace metals in mainstream cigarette smoke has been developed by introduction of an aliquot of smoke aerosol directly into an inductively coupled plasma mass spectrometer (ICP-MS) by consecutively puffing a burning cigarette. An automated apparatus consisting of a series of solenoid valves was designed and built, interfacing a smoking machine with an ICP-MS for smoke generation, collection, injection, and analysis. Air, rather than argon, was used as the sample carrier gas to minimize the temporal changes in the plasma characteristics when a discrete smoke aerosol was introduced into the plasma. The measurement precision for the system was about 2.3% RSD (n = 9) for an ambient air sample, based on the peak area of 129Xe+, in comparison with the 10–50% RSD for the analytes in the smoke matrix sample. The temporal ICP-MS signal profiles behaved differently between analytes in terms of peak growth and decay patterns, which was attributed to differences in their physical forms. For the delivery of trace metals between puffs, As, Cd, and Pb appeared to increase in the later puffs as the cigarette was smoked. In contrast, the lighting puff had more Hg than the next few puffs. The relative deliveries of As, Cd, Hg and Pb in mainstream smoke between two reference cigarettes, 1R4F and 2R4F, were compared with those measured by the conventional methods and showed good agreement. This suggests that the trace metals concentration in cigarette smoke for any test cigarettes can possibly be determined, semi-quantitatively or even quantitatively, by comparison of their signal intensities with those obtained from a reference cigarette with known trace metals concentration in its cigarette smoke.

Toxicological evaluation of glycerin as a cigarette ingredient

Glycerin is applied to cigarette tobacco at levels in the range of about 1–5% to improve moisture holding characteristics of tobacco and act as a surface active agent for flavor application. Neat material pyrolysis studies, smoke chemistry and biological activity studies (bacterial mutagenicity, cytotoxicity, in vivo micronucleus, and sub-chronic rodent inhalation) with mainstream smoke, or mainstream smoke preparations from cigarettes containing various target levels (5%, 10%, and 15%) of the glycerin were performed to provide data for an assessment of the use of glycerin as a cigarette tobacco ingredient. The actual levels of glycerin in the respective test cigarettes were determined to be 3.2%, 6.2% and 8.4% after cigarette production. At simulated tobacco burning temperatures up to 900 °C, neat glycerin did not pyrolyze extensively suggesting that glycerin would transfer intact to mainstream smoke (smoke was not analyzed for glycerin in this study). On a tar basis, nicotine in smoke was significantly decreased at 10% and 15% glycerin while water was increased at all addition levels. Addition of 10% or 15% glycerin also resulted in a statistically significant increase in acrolein (9%) and a decrease in acetaldehyde, propionaldehyde, aromatic amines, nitrogen oxides, tobacco specific nitrosamines, and phenols. Addition of 5% glycerin produced the same decrease in smoke constituents as the 10% and 15% groups but there was no concomitant increase in acrolein. Biological tests indicated no relevant differences in the genotoxic or cytotoxic potential of either mainstream smoke (or smoke preparations) from cigarettes with added glycerin compared to control cigarettes. Cigarette smoke atmosphere dilution, coupled with the lower nicotine delivery in the test cigarettes containing glycerin resulted in a lower nicotine delivery to the glycerin cigarette smoke exposed rats of the 90-day inhalation study. Smoke atmosphere acrolein was also reduced in a concentration-related manner. Incorporation of glycerin at target levels up to 15% did not produce any adverse effects in rats exposed for 90-days. The major observation in the study was a reduced biological activity of the smoke as indicated by a reduction in the severity and/or incidence of focal macrophage accumulation in the lungs and goblet cell hyperplasia/hypertrophy in the nose (level 1), and goblet cell staining depletion in the nose (level 1). The results of these studies with glycerin applied to cigarette tobacco suggest that adding glycerin to cigarette tobacco at typical use levels does not adversely alter the smoke chemistry or biological effects normally associated with exposure to mainstream cigarette smoke.

The Effect of Inhalation Volume and Breath-Hold Duration on the Retention of Nicotine and Solanesol in the Human Respiratory Tract and on Subsequent Plasma Nicotine Concentrations During Cigarette Smoking

Abstract The influence of inhalation depth and breath-hold duration on the retention of nicotine and solanesol in the human respiratory tract and on nicotine uptake was studied in ten cigarette smokers. In a first series of experiments, the subjects took seven puffs from a 10 mg ‘tar’ yield, test cigarette and a fixed volume of air (0, 75, 250, 500 or 1000 mL, as required by the protocol) was inhaled after each puff in order to give a controlled ‘depth’ of inhalation. The inhalation was drawn from a bag containing the required volume of air. Following a 2 s breath-hold, subjects exhaled normally, with the first exhalation after each puff passing through a single acidified filter pad for collection of the non-retained nicotine and solanesol. Blood samples were taken before and at intervals during and after smoking for the sessions with 0, 75 and 500 mL inhalation volumes for determination of plasma nicotine and carboxyhaemoglobin levels. Another series of experiments was conducted with a fixed inhalation volume (500 mL) and two further breath-hold durations (0 and 10 s) in addition to 2 s from above. Nicotine and solanesol retentions were measured for each breath-hold condition. The amounts of nicotine retained within the respiratory system, expressed as a percentage of the amount taken into the mouth, were consistently higher than the corresponding values for solanesol in all five inhalation conditions (0-1000 mL, 2 s breath-hold). Nicotine retention increased from 46.5% at zero inhalation to 99.5% at 1000 mL inhalation (2 s breath-hold) and from 98.0% at zero breath-hold to 99.9% at 10 s breath-hold (500 mL inhalation). Solanesol retention increased from 34.2% at zero inhalation volume to 71.9% at 1000 mL inhalation (2 s breath-hold) and from 51.8% at zero breath-hold to 87.6% at 10 s breath-hold (500 mL inhalation). Plasma nicotine decreased from pre-smoking levels after zero inhalation indicating that the nicotine retained within the mouth was poorly absorbed into the systemic circulation. After 75 mL inhalation, plasma nicotine levels were significantly greater than for zero inhalation but not significantly less than after 500 mL inhalation except at the time of maximum nicotine concentration. As in every experimental condition, a higher percentage of nicotine than solanesol was retained within the respiratory tract, it was concluded that the difference in retention of the moderately volatile nicotine and the non-volatile solanesol is consistent with the concept of nicotine evaporation from smoke particles and the subsequent efficient retention in the airways of gaseous nicotine. The retention of solanesol followed the expected pattern of particulate deposition i.e., an increase with both increasing depth of inhalation and breath-hold duration. However, nicotine retention was almost complete even at shallow inhalations and short breath-hold durations.

Toxicological evaluation of expanded shredded tobacco stems

A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Expanded shredded tobacco stems (ESS) constitute an example of a common tobacco components expansion process currently used in the manufacture of cigarettes to increase the tobacco blend filling capacity. As part of the toxicological evaluation of ESS, test cigarettes containing 9.5%, 18.5%, and 25% ESS were compared to control cigarettes containing 0% ESS. Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange), a 13-week inhalation study in Sprague–Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Collectively, data indicated that cigarettes with and without ESS had a similar toxicological profile in this test battery.

An overview of the effects of tobacco ingredients on smoke chemistry and toxicity

This paper presents an overview of a series of studies designed to assess the influence of 482 tobacco ingredients on cigarette smoke chemistry and toxicity. The studies are: pyrolysis of the ingredients; influence of the ingredients on smoke constituents believed by regulatory authorities to be relevant to smoking-related diseases (“Hoffmann analytes”); influence of the ingredients on in vitro genotoxicity and cytotoxicity of smoke partiulate matter; and influence of the ingredients on the inhalation toxicity of smoke. The present paper brings the salient features of these studies together. A pyrolysis technique has been developed which, as far as practicably possible, mimics the combustion conditions inside a burning cigarette. The results from 291 single-substance ingredients indicate that almost a third would transfer out of the cigarette burning zone at least 99% intact (i.e. less than 1% pyrolysis), and almost two thirds would transfer at least 95% intact. Of the ingredients that underwent some degree of pyrolysis, a few “Hoffmann analytes” were detected amongst the pyrolysis products of 19 ingredients. Taking into account maximum use levels, their maximum pyrolysis levels were generally small and often insignificant compared to the levels typically present in smoke. Possible exceptions were acetaldehyde and benzene from the pyrolysis of malic acid. However, subsequent smoke chemistry studies indicated that the maximum levels predicted from pyrolysis of this involatile substance were overestimated, suggesting that malic acid does not undergo complete pyrolysis in the burning cigarette and/or generates acetaldehyde and benzene at similar rates to that of tobacco on a per weight basis. When added to tobacco, many of the ingredient mixtures produced no significant effect on the levels of many of the “Hoffmann analytes” in smoke, while some produced increases or decreases relative to the relevant control cigarettes. The study has concentrated on the increases. Many of the differences were found to be not significant when the long-term variability of the analytical methodology was taken into account. However, even taking this into account, the smoke formaldehyde levels in two of the test cigarettes were significantly increased relative to their controls, by up to 26 μg (73%). These increases are likely to be due to the pyrolysis of sugars, cellulose and other polysaccharide materials. The activity of smoke particulate matter from cigarettes containing tobacco ingredients has been determined with three in vitro bioassays, two for genotoxicity and one for cytotoxicity. These were the Ames test, the mammalian cell micronucleus assay, and the neutral red uptake cytotoxicity assay. Within the sensitivity and specificity of these bioassays, the specific activity of the cigarette smoke particulate matter was not changed by the addition of ingredients to the cigarette. Three 90-day sub-chronic inhalation studies have been undertaken and histopathological and histomorphometric assessments made within the respiratory tracts of animals exposed to smoke from cigarettes containing the various ingredient mixtures and their control cigarettes. The response due to tobacco smoke exposure was not distinguishable between the test and control cigarettes, indicating that the presence of the ingredients had made no discernable differences to the type and severity of the treatment-related changes.

A comparison of in vitro toxicities of cigarette smoke condensate from Eclipse cigarettes and four commercially available ultra low-“tar” cigarettes

Eclipse is a cigarette that primarily heats rather than burns tobacco. R.J. Reynolds Tobacco Company (RJRT) has previously reported the results of in vitro toxicity studies comparing Eclipse with University of Kentucky 1R5F and 1R4F reference cigarettes. To characterize the differences between Eclipse and very low yielding/ultra low-“tar” (vULT) tobacco-burning cigarettes, RJRT conducted a comparative evaluation of the genotoxicity and cytotoxicity of mainstream cigarette smoke condensate (CSC) from Eclipse and three vULT tobacco-burning cigarettes (Now 83 Box, Merit Ultima and Carlton Soft Pack) as well as the leading ultra low-“tar” (ULT) brandstyle (Marlboro Ultra Lights) under four smoking regimens: (1) FTC—35 ml puff volume every 60 s for a 2 s duration (35/60/2); (2) 50/30/2, 0% vents blocked; (3) Massachusetts—45/30/2, 50% vents blocked; (4) Canadian—55/30/2, 100% vents blocked. Ames testing indicated that Eclipse CSC was less ( P<0.05) mutagenic than CSC from the four cigarettes under all smoking regimens when compared on a revertants per mg Total Particulate Matter (TPM) basis. When mutagenicity was calculated on a revertants per cigarette basis the mutagenicity of Eclipse CSC was lower ( P<0.05) than the mutagenicity of Merit Ultima, Carlton Soft Pack, and Marlboro Ultra Lights, regardless of the puffing regimen. On a per cigarette basis, the calculated mutagenicity of Eclipse was higher ( P<0.05) than Now 83 Box cigarettes in the FTC and 50/30/2 regimens but lower ( P<0.05) in the Massachusetts and Canadian regimens. Eclipse CSC was less ( P<0.05) cytotoxic as measured in the neutral red assay (based on EC 50 values—μg TPM/ml media) than the CSC from the four test cigarettes regardless of the regimen used. Collectively, these data demonstrate that the toxicity of CSC from Eclipse is significantly reduced relative to the activity of CSC from the tested vULT cigarettes and the Marlboro Ultra Lights.

The effect of tobacco ingredients on smoke chemistry. Part I: Flavourings and additives

The effects of 450 tobacco ingredients added to tobacco on the forty-four “Hoffmann analytes” in mainstream cigarette smoke have been determined. These analytes are believed by regulatory authorities in the USA and Canada to be relevant to smoking-related diseases. They are based on lists published by D. Hoffmann and co-workers of the American Health Foundation in New York. The ingredients comprised 431 flavours, 1 flavour/solvent, 1 solvent, 7 preservatives, 5 binders, 2 humectants, 2 process aids and 1 filler. The cigarettes containing mixtures of the ingredients were smoked using the standard ISO smoking machine conditions. The levels of the “Hoffmann analytes” in the smoke from the test cigarettes containing the ingredient mixture were compared to those from control cigarettes without the ingredients. In practice, flavouring ingredients are typically added to tobacco that also contains casing ingredients and reconstituted tobacco materials. In order to keep the tobacco mixtures as authentic as possible, three comparisons have been made in this study. These are: (a) control cigarette containing a typical US blended, cased tobacco incorporating reconstituted tobacco versus test cigarettes that had flavouring ingredients added to this tobacco; (b) control cigarette containing tobacco only versus test cigarettes with the tobacco cased and incorporating flavourings; (c) control cigarette containing tobacco only versus test cigarette incorporating additives made in an experimental sheet material. The significances of differences between the test and control cigarettes were determined using both the variability of the data on the specific occasion of the measurement, and also taking into account the long-term variability of the analytical measurements over the one-year period in which analyses were determined in the present study. This long-term variability was determined by measuring the levels of the 44 “Hoffmann analytes” in a reference cigarette on many occasions over the one-year period of this study. The ingredients were added to the experimental cigarettes at or above the maximum levels used commercially by British American Tobacco. The effect of the ingredient mixtures on total particulate matter and carbon monoxide levels in smoke was not significantly different to the control in most cases, and was never more than 10% with any ingredient mixture. It was found that, in most cases, the mixtures of flavouring ingredients (generally added in parts per million levels) had no statistically significant effect on the analyte smoke yields relative to the control cigarette. Occasionally with some of the mixtures, both increases and decreases were observed for some smoke analyte levels relative to the control cigarette. These differences were generally up to about 15% with the mixtures containing flavouring ingredients. The significance of many of the differences was not present when the long-term variability of the analytical methodology was taken into account. For the test cigarettes with ingredient mixtures containing casing ingredients, there were again no significant changes in smoke analyte levels in most cases. Those changes that were observed are as follows. Decreases in smoke levels were observed with some ingredient mixtures for most of the tobacco specific nitrosamines (up to 24%), NO x , most of the phenols (up to 34%), benzo[ a]pyrene, and some of the aromatic amines and miscellaneous organic compounds on the “Hoffmann list”. Increases were observed for some test cigarettes in smoke ammonia, HCN, formaldehyde and lead levels (up to 24%). The significance of the ammonia and lead increases was not present when the long-term variability of the analytical methodology was taken into account. The yields of some carbonyl compounds in smoke were increased in one comparison with an additives mixture containing cellulosic components; in particular, formaldehyde was increased by 68%. This was the largest single change seen in any smoke analyte level in this study. These carbonyls are produced from the pyrolysis of cellulosic and other polysaccharide materials, present in the additives mixture. With this test cigarette, all tobacco specific nitrosamines, phenols, semi-volatile bases, NO x and some aromatic amines and miscellaneous organic compounds on the “Hoffmann list” were decreased, by up to 22%. The significance of many of these differences remained even when the long-term variability of the analytical methodology was taken into account. The levels of all other “Hoffmann analytes” in the smoke were not significantly different to those of the control cigarette. With the exception of the determinations of “tar”, nicotine and carbon monoxide, there are currently no internationally recognised standard methods for measurement of the other “Hoffmann analytes”. Each laboratory uses its own methods and there are large laboratory-to-laboratory variations, as well as variations over time in a given laboratory. Therefore, it is important that in any comparison of smoke analytes amongst different cigarettes, all the analytes should be measured in the same laboratory and at the same time. This was the case in the present study and all the methods have been validated internally.

Toxicological evaluation of propane expanded tobacco

A tiered testing strategy has been developed to evaluate the potential for tobacco processes, ingredients, and other technological developments to increase or decrease the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Propane expanded tobacco is an example of a processed tobacco used in the modern manufacture of cigarettes. Test cigarettes containing propane expanded tobacco were compared to control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11). The toxicological evaluation included chemistry studies using mainstream cigarette smoke (determination of selected constituent yields), in vitro studies using cigarette smoke condensate (Ames study in Salmonella typhimurium and sister chromatid exchange study in Chinese hamster ovary cells) and in vivo studies (13-week inhalation study of mainstream cigarette smoke in Sprague–Dawley rats and 30-week dermal tumor promotion study of cigarette smoke condensate in SENCAR mice). Although statistically significant differences in several smoke constituents were observed, most constituents from cigarettes containing 100% propane expanded tobacco were within market survey ranges. Furthermore, biological tests indicated that the cigarettes containing propane or Freon-11 expanded tobacco were not significantly different.

Toxicological evaluation of dry ice expanded tobacco

A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Dry ice expanded tobacco (DIET) is an example of a common tobacco expansion process currently used in the manufacture of cigarettes to increase tobacco filling capacity. As part of the toxicological evaluation of DIET, test cigarettes containing DIET were compared with control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11, also known as trichlorofluoromethane). Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange, SCE), a 13-week inhalation study in Sprague–Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Cigarettes containing DIET or Freon-11 expanded tobacco were similar in biological activity.

Bacillus thuringiensis (Bt) for the Control of Insect Pests in Stored Tobacco: A Review

Abstract Among the insect species causing infestations and serious damages to stored commodities, the cigarette beetle, Lasiodermaserricorne (F.) and the tobacco moth, Ephestiaelutella (Hübner) are the major pests of both raw and manufactured tobacco. Post-harvest tobacco control is achieved through sanitation, insect monitoring, and fumigation with phosphine. However, insect resistance to phosphine and control failures have been reported, and increasing regulatory pressure is being exerted on fumigants. Biological control agents such as Bacillus thuringiensis (Bt) appear to be environmentally sound and potentially viable alternatives to chemical control. Bt is a bacterium that produces insecticidal crystal proteins during the sporulation phase and has been, for more than 40 years, the microorganism of choice for the biocontrol of phytophagous insect pests. It produces insecticidal crystal proteins that display specific activity against certain orders of insects and become active upon ingestion by the insect. Our laboratory has conducted extensive research and worldwide surveys to evaluate the presence of Bt in stored tobacco and has confirmed previous findings indicating that Bt may be considered part of the naturally occurring phylloplanemicroflora. Several Bt strains were isolated from tobacco and characterized by DNA and protein profiling. The insecticidal activity of selected strains and of two commercial products against the larvae of L. serricorne was determined by diet incorporation assays. Moreover, the stability of Bt spores and crystal proteins on cured tobacco leaves was assessed over a storage period of time of 30 months. Cigarette prototypes were made with Bt-treated tobacco. Standard cigarette and smoke evaluations did not show any significant difference between the test and control cigarettes. Although the tested Bt strains and products did not yield satisfactory levels of mortality at the required times and doses, the experimental results summarized in the present review indicate that B. thuringiensishas potential for the control of the cigarette beetle. The integration of conventional control approaches with novel systems based on biological agents with different modes of action should offer new avenues for the effective management of stored tobacco pests in line with integrated pest management (IPM) concepts.

TOXICOLOGIC EVALUATION OF HUMECTANTS ADDED TO CIGARETTE TOBACCO: 13-WEEK SMOKE INHALATION STUDY OF GLYCERIN AND PROPYLENE GLYCOL IN FISCHER 344 RATS

Glycerin (CAS no. 56-81-5) and propylene glycol (CAS no. 57-55-6) are commonly used as humectant ingredients in manufactured cigarettes to control and maintain the moisture content of the cut tobacco filler. The potential of these added humectants to affect the toxicity of cigarette smoke was investigated in a subchronic nose-only smoke inhalation study in rats. Filtered test cigarettes were prepared from an American-style tobacco blend containing either glycerin added at 5.1% w/w tobacco, propylene glycol at 2.2% w/w tobacco, or combinations of these humectants totaling 2.3%, 3.9%, and 7.2% w/w tobacco. Other groups of animals were exposed similarly to the smoke of reference cigarettes without added humectants, or to filtered air (sham control). Smoke exposures were conducted for 1 h/ day, 5 days/wk for 13 wk, at target smoke particulate concentrations of 350 mg/m 3. All smoke-exposed groups had equivalent increases in blood carboxyhemoglobin, serum nicotine, and serum cotinine relative to the air controls. Smoke-associated reductions in body weights and occasional increases in heart and lung weights were generally similar among the various exposure conditions at necropsy. Increases in serum alkaline phosphatase and decreases in serum glucose and cholesterol were observed among smoke-exposed females relative to air controls. However, no significant differences in these parameters were evident between the humectant-containing and reference cigarette smoke exposure groups. Assessments of respiration conducted after 3, 6, 9, and 12 wk of smoke exposure indicated an initial smoke-related but not humectant-related decrease in respiratory rate, tidal volume, and minute volume during the first 20 min of each smoke exposure. Respiratory-tract histopathology was consistent across sexes and smoke groups, comprising (1) diffuse and focal alveolar pigmented macrophages and chronic interstitial inflammation in the lung, (2) laryngeal epithelial hyperplasia, squamous metaplasia, and scab formation, and (3) epithelial hyperplasia in the anterior nose. Smoke-related histopathology resolved substantially during a 6-wk postexposure recovery period. Addition of the tested humectants to cigarettes, singly or in combination, had no meaningful effect on the site, occurrence, or severity of respiratory-tract changes or on the measured indices of pulmonary function. It was concluded that the addition of glycerin and propylene glycol to cigarettes does not significantly affect the biological activity of inhaled cigarette smoke in this rat model.

Evaluation of the potential effects of ingredients added to cigarettes. Part 1: Cigarette design, testing approach, and review of results

A testing program was designed to evaluate the potential effects of 333 ingredients added to typical commercial blended test cigarettes on selected biological and chemical endpoints. Ingredients were incorporated into the test cigarettes as they are normally used in the manufacturing process. The studies performed included a bacterial mutagenicity screen (Ames assay), a mammalian cell cytotoxicity assay (neutral red uptake), determination of smoke chemical constituents, and a 90-day nose-only smoke inhalation study in rats. Three pairs of test cigarettes were produced, each containing one of three different groups of ingredients. In each pair, one of the cigarettes contained the normal approximate use level of the ingredients (low-level) and the other a 1.5–3 multiple of the normal use level (high-level). Analysis of the test cigarettes for selected ingredients or markers indicated that the target application rates were achieved and that the cigarettes had been manufactured as intended. Evaluation of cigarette performance indicated that the addition of the ingredients at high levels did not significantly alter the burning characteristics of the test cigarettes. Specific details of the individual studies conducted as part of an ingredient evaluation program are discussed in Parts 2–4 of this publication series (Food and Chemical Toxicology, 2002, 40, 93–104; Food and Chemical Toxicology, 2002, 40, 105–111; Food and Chemical Toxicology, 2002, 40, 113–131). The results of the smoke chemistry studies indicated a reduction in the majority of the smoke constituents and a few isolated instances of increases when compared to the control cigarettes. These smoke chemistry changes, while statistically significant, were not supported by any significant alteration in the biological effects of cigarette smoke normally seen with the bacterial mutagenicity assay, cytotoxicity assay or subchronic inhalation study. Based on the results of these studies, it can be concluded that these ingredients added to tobacco do not add significantly to the overall toxicity of cigarettes.

Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity

Mainstream smoke from blended research cigarettes with (test) and without (control) the addition of ingredients to the tobacco was assayed for inhalation toxicity. In total, 333 ingredients commonly used in cigarette manufacturing were assigned to three different groups. Each group of ingredients was introduced at a low and a high level to the test cigarettes. Male and female Sprague–Dawley rats were exposed nose-only either to fresh air (sham) or diluted mainstream smoke from the test, the control, or the Reference Cigarette 1R4F at a concentration of 150 μg total particulate matter/l for 90 days, 6 h/day, 7 days/week. A 42-day post-inhalation period was included to evaluate reversibility of possible findings. There were no remarkable differences in in-life observations or gross pathology between test and control groups. An increase in activity of liver enzymes, known to be due to the high smoke dose, revealed no toxicologically relevant differences between the test and control groups. No toxicological differences were seen between the test and control groups for smoke-related hematological changes, such as a decrease in total leukocyte count. The basic smoke-related histopathological effects, which were more pronounced in the upper respiratory tract than in the lower respiratory tract, were hyperplasia and squamous metaplasia of the respiratory epithelium, squamous metaplasia and atrophy of the olfactory epithelium, and accumulation of pigmented alveolar macrophages. There were no relevant qualitative or quantitative differences in findings in the respiratory tract of the rats exposed to the smoke from the control and test cigarettes. The data indicate that the addition of these 333 commonly used ingredients, added to cigarettes in three groups, did not increase the inhalation toxicity of the smoke, even at the exaggerated levels used.

Evaluation of the potential effects of ingredients added to cigarettes. Part 2: Chemical composition of mainstream smoke

Cigarette mainstream smoke from blended research cigarettes with and without the addition of ingredients was analyzed for its chemical composition. In total, 333 ingredients commonly used in cigarette manufacturing were assigned to three different groups. Each group of ingredients was introduced at a low and a high level to the test cigarettes. The list of the 51 smoke constituents determined is based on those analytes suggested for analysis in a US Consumer Product Safety Commission proposal for low ignition cigarettes and cigarette smoke constituents identified by the International Agency for Research on Cancer as worthy of concern and characterized as carcinogens. An increase in the yield of total particulate matter (TPM) in the range of 13 to 28% relative to the control cigarette without ingredients was observed for all test cigarettes. This was presumably caused by the higher transfer rates of the added ingredients to the smoke compared to the transfer from the tobacco part of the filler. When the yields of individual constituents were normalized to the TPM yields, a reduction in the majority of the constituents was observed when compared to the control. For one of the ingredient groups this reduction was especially high: for phenols a maximum of 70%, for polycyclic aromatic hydrocarbons 50%, and for N-nitrosamines 45%. An increase in the amount relative to TPM was observed for a few smoke constituents: hydrogen cyanide and cadmium (one ingredient group), formaldehyde (one ingredient group), and resorcinol and lead (two ingredient groups). These results are consistent with the lack of any increased activity in the in vitro and in vivo assays in this same series of studies (Food and Chemical Toxicology 2002, 40, 105–111; Food and Chemical Toxicology 2002, 40, 113–131). An overall assessment of our data suggests that these ingredients, when added to the tobacco, do not add to the toxicity of smoke, even at the elevated levels tested in this series of studies.

Evaluation of the potential effects of ingredients added to cigarettes. Part 3: In vitro genotoxicity and cytotoxicity

Cigarette mainstream smoke from blended cigarettes with and without the addition of ingredients was assayed for its cytotoxicity and genotoxicity. In total, 333 ingredients commonly used in cigarette manufacturing were assigned to three different groups. Each group of ingredients was added at a low and a high level to the test cigarettes. The mutagenicity of the particulate phase of the resulting cigarette smoke was assayed in the Salmonella plate incorporation (Ames) assay with tester strains TA98, TA100, TA102, TA1535 and TA1537. The cytotoxicity of the gas/vapor phase and the particulate phase was determined in the neutral red uptake assay with mouse embryo BALB/c 3T3 cells. Within the sensitivity and specificity of the test systems, the in vitro mutagenicity and cytotoxicity of the cigarette smoke were not increased by the addition of the ingredients.

Toxicologic evaluation of flavor ingredients added to cigarette tobacco: skin painting bioassay of cigarette smoke condensate in SENCAR mice

Four comparative two-stage SENCAR mouse skin painting bioassays were conducted with cigarette smoke condensate (CSC) preparations to evaluate the effect of common American cigarette flavoring ingredients on tumor promotion. Each independent study employed a unique flavoring combination applied to tobacco at exaggerated levels, and in total resulted in an evaluation of 150 ingredients. Groups of 30–50 female SENCAR mice each were initiated topically with 50 μg of 7,12-dimethylbenz(a)anthracene (DMBA), and promoted thrice weekly for 26 weeks with either 10 or 20 mg of CSC from test cigarettes containing ingredient mixtures. For comparison, separate groups of mice received concurrent treatment with CSC from reference cigarettes prepared without added ingredients. Negative and positive controls were treated with acetone or 12-0-tetradecanoyl-phorbol-13-acetate (TPA) as a promoter, respectively. CSC-only groups served as promotion controls. Tumors developed in >80% of the TPA-treated mice by study week 11, with a <3% background tumor formation in the acetone treated controls at termination. Tumor incidence in CSC-only promotion control groups was <20%, with no apparent difference between reference and test CSC groups. Approximately 70% of the DMBA-initiated mice promoted with 20 mg CSC developed tumors. Tumors first appeared around week 9, with about five tumors/tumor bearing animal. Tumor incidence, latency and multiplicity were CSC dose related, with a lower tumor incidence (approximately 50%), longer latency (12 weeks), and reduced tumor burden (four tumors/tumor bearing animal) at the 10 mg CSC dose level. While tumor incidence, latency and multiplicity data occasionally differed between test and comparative reference CSC groups, all effects appeared to be within normal variation for the model system. Furthermore, none of the changes appeared to be substantial enough to conclude that the tumor promotion capacity of CSC obtained from cigarettes containing tobacco with ingredients was discernibly different from the CSC obtained from reference cigarettes containing tobacco processed without ingredients.

Smoking and airway hyperresponsiveness especially in the presence of blood eosinophilia increase the risk to develop respiratory symptoms: a 25-year follow-up study in the general adult population.

Airway hyperresponsiveness (AHR) constitutes a risk for development of respiratory symptoms. We assessed whether blood eosinophilia (>/= 275 eosinophils/microliters), skin test positivity (sum score >/= 3) and cigarette smoking (never, ex-smoker, 1-14 cig/d, 15-24 cig/d, >/= 25 cig/d) at the first of two successive surveys are related to the development of respiratory symptoms (chronic cough or phlegm, bronchitis, persistent wheeze, dyspnea, and asthma) at the second survey, and whether these relations are the same in subjects with (PC10 </= 8 mg/ml histamine) and without AHR. We analyzed data of the longitudinal Vlagtwedde-Vlaardingen Study (1965 to 1990) using logistic regression analyses with paired observations, taking multiple measurements within a person into account. In total, 995 men and 792 women contributed 4,403 paired observations. Eosinophilia in hyperresponsive subjects significantly increased the risk to develop one or more respiratory symptoms (odds ratio [OR] = 3.67, 95% confidence interval [CI] = 1.79 to 7.52), wheeze (OR = 5. 06, 95% CI = 2.11 to 12.13), and dyspnea (OR = 2.73, 95% CI = 1.13 to 6.60), independent of smoking, age, sex, area of residence, and time between two successive surveys. Smoking at the first of two successive surveys increased the risk to develop symptoms in a dose-dependent relation. Subjects with positive skin tests in the past were less likely to develop one or more respiratory symptoms (OR = 0.64, 95% CI = 0.46 to 0.88) and chronic phlegm (OR = 0.65, 95% CI = 0.42 to 1.00), independent of AHR. This longitudinal study in the general adult population shows that cigarette smoking and hyperresponsive subjects are at increased risk to develop respiratory symptoms, and especially so when eosinophilia is present in hyperresponsive persons.

Comparison of the propensity of cigarettes to ignite upholstered furniture fabrics and cotton ducks (500-fabric study)? a second opinion

In the paper entitled ‘Comparison of the Propensity of Cigarettes to Ignite Upholstered Furniture Fabrics and Cotton Ducks (500-Fabric Study)’ (Fire Mater. 21, 123–141 (1997)) Marcelo M. Hirschler addresses the validity of a test method, proposed by NIST, for smoldering cigarette ignition propensity of upholstery fabrics. The thrust of the study is to establish similarities ‘between the ignition propensity of cigarettes assessed by (1) a set of 500-upholstery fabrics (chosen at random among typical upholstery fabrics) and (2) a test method proposed by NIST (NIST 851) and based on “cotton duck” fabrics’. The conclusion of Hirschler's study is that ‘the overall results obtained from the 500-upholstery fabric study correlate well with those of the “cotton duck” study.’ In addition, the author states that ‘the “cotton duck” can be considered, as a whole, to behave similarly to the majority (estimated at perhaps 80%) of the upholstery fabrics available at the time of the study, and the test is valid’. In an attempt to validate these statements, the ignition patterns generated by the five test cigarettes on each of the 500-upholstery fabrics were compared with the NIST “cotton duck” pattern. Only 6.6% were found to generate a pattern similar to the NIST pattern and 94% of this group were heavyweight (greater than 14 oz/sq yd.) fabrics. Assessment of the test results fails to substantiate Hirschler's statements. © 1998 John Wiley & Sons, Ltd.

Comparative Cytotoxicity Studies of Smoke Condensates from Different Types of Cigarettes and Tobaccos

The neutral red assay, a rapid and accurate method for estimating the cytotoxicity of chemicals, has been used to assess the cytotoxicity of cigarette smoke condensate (CSC), a complex chemical mixture containing over 3000 identified compounds. The first objective was to optimize the neutral red assay for evaluation of CSCs. This study also assessed and compared the cytotoxicity of smoke condensates from three reference cigarettes which differ in ‘tar’ content; cigarettes of different tobacco type composition; an ultra-low tar cigarette (R1); and an RJR test cigarette which heats but does not burn tobacco. Finally, this study investigated the cytotoxicity of a specific CSC component, nicotine, and its metabolite, cotinine. Exposure times of 24 hours or longer using CHO cells provided optimal conditions for evaluation of CSC cytotoxicity. The cytotoxicity of CSCs from reference cigarettes was similar. CSC from cigarettes comprised of flue-cured tobacco exhibited greater cytotoxicity than CSC from cigarettes comprised of burley tobacco. CSC from the R1 cigarette exhibited similar cytotoxicity compared with 1R4F CSC. The CSC from a cigarette that heated but did not burn tobacco (RJR test cigarette) demonstrated no cytotoxicity in CHO cells. Finally, nicotine and cotinine were not cytotoxic to CHO cells. The neutral red assay has been proved useful for quantifying differences in cytotoxicity of smoke condensates from cigarettes which vary in ‘tar’ yield and for assessing specific smoke constituents.

Chemical and Biological Studies of a New Cigarette that Primarily Heats Tobacco.Part 3. In Vitro Toxicity of Whole Smoke

Mainstream smoke from Kentucky reference low “tar” (1R4F) and ultra-low “tar” (1R5F) cigarettes and a test cigarette (TOB-HT), that primarily heats tobacco, was compared for cytotoxic and genotoxic potential using cellular smoke exposure technology (CSET). CSET includes a computer controlled 30-port AMESA/Battelle-Geneva smoke generator which exposes cultured mammalian Chinese hamster ovary cells (CHO) to whole smoke. Cytotoxicity was assessed using the neutral red assay and genotoxicity was assessed using the sister chromatid exchange (SCE) assay. Compared on a per cigarette basis, mainstream smoke from 1R5F and the TOB-HT cigarette was significantly less cytotoxic and genotoxic than the smoke from the 1R4F cigarette. The cytotoxic and genotoxic activity of smoke from the TOB-HT cigarettes was slightly greater than the smoke from the ultra-low “tar” Kentucky 1R5F reference cigarettes. In conclusion, in these assays mainstream whole smoke of the TOB-HT cigarette had slightly greater cytotoxic and genotoxic potential compared with an ultra-low “tar” 1R5F Kentucky reference cigarette and significantly less activity compared with the whole mainstream smoke from a low “tar” 1R4F Kentucky reference cigarette, representative of the US market average cigarette for FTC yields of “tar”, CO and nicotine.