Toxicological evaluation of an electrically heated cigarette. Part 3: Genotoxicity and cytotoxicity of mainstream smoke

The in vitro toxicity of cigarette mainstream smoke from an electrically heated cigarette (EHC) with controlled combustion was compared with that of the standard University of Kentucky Reference Cigarette 1R4F.In the Salmonella reverse mutation assay, strains TA98, TA100, TA102, TA1535 and TA1537 were used in the absence and presence of a metabolic promutagen activation system (S9) to determine the mutagenic potential of the total particulate matter (TPM), which was collected on a glass-fiber filter. In the neutral red uptake assay, mouse embryo BALB/c 3T3 cells were used to determine the cytotoxic potential of TPM as well as of the water-solubles in the gas/vapor phase trapped in phosphate-buffered saline.The TPM from the electrically heated cigarette was up to 90% lower in mutagenicity than that of the 1R4F calculated on an equal TPM basis. This reduction in mutagenicity is consistent with the significantly lower concentration of nearly all constituents analyzed in EHC smoke. With regard to cytotoxicity when calculated on an equal TPM basis, TPM from the electrically heated cigarette was 40% less active relative to the 1R4F. When calculated on a per cigarette basis, the cytotoxicity of both the TPM fraction and the water-solubles in the gas/vapor phase of smoke from the EHC was ca. 80% lower relative to the 1R4F.     

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.     

Effect of pyrolysis temperature on the mutagenicity of tobacco smoke condensate.

Tobacco smoke aerosols with fewer mutagens in the particulate fraction may present reduced risk to the smoker. The objective of this study was to test the hypothesis that the temperature at which tobacco is pyrolyzed or combusted can affect the mutagenicity of the particulate fraction of the smoke aerosol. Tobacco smoke aerosol was generated under precisely controlled temperature conditions from 250 to 550 degrees C by heating compressed tobacco tablets in air. The tobacco aerosols generated had a cigarette smoke-like appearance and aroma. The tobacco smoke aerosol was passed through a Cambridge filter pad to collect the particulate fraction, termed the smoke condensate. Although condensates of tobacco smoke and whole cigarette mainstream smoke share many of the same chemical components, there are physical and chemical differences between the two complex mixtures. The condensates from smoke aerosols prepared at different temperatures were assayed in the Ames Salmonella microsome test with metabolic activation by rat liver S9 using tester strains TA98 and TA100. Tobacco smoke condensates were not detectably mutagenic in strain TA98 when the tobacco smoke aerosol was generated at temperatures below 400 degrees C. Above 400 degrees C, condensates were mutagenic in strain TA98. Similarly, condensates prepared from tobacco smoke aerosols generated at temperatures below 475 degrees C were not detectably mutagenic in strain TA100. In contrast, tobacco tablets heated to temperatures of 475 degrees C or greater generated smoke aerosol that was detectably mutagenic as measured in TA100. Therefore, heating and pyrolyzing tobacco at temperatures below those found in tobacco burning cigarettes reduces the mutagenicity of the smoke condensate. Highly mutagenic heterocyclic amines derived from the pyrolysis of tobacco leaf protein may be important contributors to the high temperature production of tobacco smoke Ames Salmonella mutagens. The relevance of these findings regarding cancer risk in humans is difficult to assess because of the lack of a direct correlation between mutagenicity in the Ames Salmonella test and carcinogenicity.     

Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter

A novel carbon filter has been developed which primarily reducesthe amount of certain vapor phase constituents of tobacco smokewith greater efficiency than the charcoal filters of cigarettescurrently in the market In vitro indicators of genotoxic andcytotoxic potential were used to compare the cigarette smokecondensate (particulate phase) or whole cigarette smoke (vaporphase and particulate phase) from cigarettes containing thenovel carbon filter with smoke condensate or whole smoke fromcommercial or prototype cigarettes not containing the novelcarbon filter. Ames bacterial mutagenicity, sister chromatidexchange (SCE) in Chinese hamster ovary (CHO) cells, and neutralred cytotoxicity assays in CHO cells were utilized to assessthe genotoxic and cytotoxic potential of the cigarette smokecondensates. SCE and neutral red cytotoxicity assays were utilizedto assess the genotoxic and cytotoxic potential of the wholesmoke. As expected, the novel carbon filter did not significantlyaffect the genotoxic or cytotoxic activity of the smoke condensate,although we did observe that the use of low-nitrogen tobaccoreduced the mutagenicity of the condensate in Salmonella typhimuriumstrain TA98. However, the whole smoke from cigarettes containingthe novel carbon filter demonstrated significant reductionsin genotoxic and cytotoxic potential compared to cigaretteswithout the novel carbon filter. The toxicity of the smoke wascorrelated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCEinduction) to the aggregate mass of several vapor phase components(acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene,ammonia, NO_x, HCN, benzene, isoprene, and formaldehyde) in thesmoke of the cigarettes utilized in this study. In conclusion,this novel carbon filter, which significantly reduced the amountof carbonyls and other volatiles in mainstream cigarette smoke,resulted in significant reductions in the genotoxic and cytotoxicactivity of the smoke as measured by these assays.     

Toxicological considerations on the use of propylene glycol as a humectant in cigarettes

Propylene glycol (PG) is a humectant commonly used in cigarettes. Previous toxicological examinations of the effects on the addition of PG to tobacco used mixtures with several other flavoring agents. In the present work, toxicological comparisons were made of experimental cigarettes containing no added PG against otherwise similar cigarettes with three different amounts of PG added to the tobacco. The main toxicological comparison was a sub-chronic inhalation study with mainstream smoke in Sprague–Dawley rats (exposures of 150 mg/m³ of total particulate matter, 6 h exposure per day, for 90 consecutive days). The target PG concentrations in the tobacco of the four cigarette types were 0, 4, 7 and 10%. Additional studies with mainstream smoke were bacterial mutagenicity (5 Salmonella strains, both with and without metabolic activation, particulate phase only), cytotoxicity of both particulate and gas/vapor phases (using the neutral red uptake assay), and analytical chemistry (41 analytes). The graded inclusion of PG into experimental cigarettes resulted in increases in the smoke concentrations of propylene oxide, at very low concentrations. Broadly similar responses were seen across the four cigarette types, and the responses were similar to those previously described in the scientific literature. The addition of PG to experimental cigarettes reduced concentrations of some smoke components (e.g. nicotine), but had minimal effects on the biological responses. Most of the changes produced in the 90-days of exposure were resolved in a 42-day post-inhalation period.     

Toxicological comparisons of cigarettes containing different amounts of vanillin

Vanillin is a flavoring agent used in cigarettes. Previous toxicological examinations of the effects on the addition of vanillin to tobacco used mixtures with several other flavoring agents. In the present work, toxicological comparisons were made of experimental cigarettes containing no added vanillin against otherwise similar cigarettes with three different amounts of vanillin added to the tobacco. The main toxicological comparison was a subchronic inhalation study with mainstream smoke in Sprague-Dawley rats (exposures of 150 mg/m3 of total particulate matter, 6 h exposure per day, for 90 consecutive days). Vanillin concentrations in the tobacco of the 4 cigarette types at the end of the study were 0, 67, 1233, and 3109 ppm. Additional studies with mainstream smoke were Salmonella mutagenicity (5 bacterial strains, both with and without metabolic activation, particulate phase only), cytotoxicity of both particulate and gas/vapor phases (using the neutral red uptake assay), and analytical chemistry (49 analytes, including 5 metals). Similar responses were seen across the four cigarette types, and the responses were similar to those previously described in the scientific literature. At the same smoke concentration, the inhalation exposures produced effectively the same responses, in each of the four groups. Most of the changes produced in the 90 days of exposure were resolved in a 42-day postinhalation period. The addition of vanillin to tobacco at inclusion rates up to 3109 ppm did not influence a broad range of toxicological endpoints.     

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.     

Toxicological Comparisons of Cigarettes Containing Different Amounts of Vanillin

Vanillin is a flavoring agent used in cigarettes. Previous toxicological examinations of the effects on the addition of vanillin to tobacco used mixtures with several other flavoring agents. In the present work, toxicological comparisons were made of experimental cigarettes containing no added vanillin against otherwise similar cigarettes with three different amounts of vanillin added to the tobacco. The main toxicological comparison was a subchronic inhalation study with mainstream smoke in Sprague-Dawley rats (exposures of 150 mg/m³ of total particulate matter, 6 h exposure per day, for 90 consecutive days). Vanillin concentrations in the tobacco of the 4 cigarette types at the end of the study were 0, 67, 1233, and 3109 ppm. Additional studies with mainstream smoke were Salmonella mutagenicity (5 bacterial strains, both with and without metabolic activation, particulate phase only), cytotoxicity of both particulate and gas/vapor phases (using the neutral red uptake assay), and analytical chemistry (49 analytes, including 5 metals). Similar responses were seen across the four cigarette types, and the responses were similar to those previously described in the scientific literature. At the same smoke concentration, the inhalation exposures produced effectively the same responses, in each of the four groups. Most of the changes produced in the 90 days of exposure were resolved in a 42-day postinhalation period. The addition of vanillin to tobacco at inclusion rates up to 3109 ppm did not influence a broad range of toxicological endpoints.     

The mouse lymphoma thymidine kinase assay for the assessment and comparison of the mutagenic activity of cigarette mainstream smoke particulate phase

The mouse lymphoma thymidine kinase assay (MLA) has been optimized to quantitatively determine the in vitro mutagenicity of cigarette mainstream smoke particulate phase. To test whether the MLA is able to discriminate between different cigarette types, specially constructed cigarettes each containing a single tobacco type - Bright, Burley, or Oriental - were investigated. The mutagenic activity of the Burley cigarette was statistically significantly lower, up to approximately 40%, than that of the Bright and Oriental cigarettes. To determine the impact of two different sets of smoking conditions, American-blend cigarettes were smoked under US Federal Trade Commission/International Organisation for Standardisation conditions and under Massachusetts Department of Public Health (MDPH) conditions. Conventional cigarettes - eight from the US commercial market plus the Reference Cigarettes 1R4F and 2R4F - and an electrically heated cigarette smoking system (EHCSS) prototype were tested. There were no statistically significant differences between the two sets of smoking conditions on a per mg total particulate matter basis, although there was a consistent trend towards slightly lower mutagenic activity under MDPH conditions. The mutagenic activity of the EHCSS prototype was distinctly lower than that of the conventional cigarettes under both sets of smoking conditions. These results show that the MLA can be used to assess and compare the mutagenic activity of cigarette mainstream smoke particulate phase in the comprehensive toxicological assessment of cigarette smoke.     

Effects of flavoring and casing ingredients on the toxicity of mainstream cigarette smoke in rats

A series of in vitro and in vivo studies evaluated the potential effects of tobacco flavoring and casing ingredients. Study 1 utilized as a reference control cigarette a typical commercial tobacco blend without flavoring ingredients, and a test cigarette containing a mixture of 165 low-use flavoring ingredients. Study 2 utilized the same reference control cigarette as used in study 1 and a test cigarette containing eight high-use ingredients. The in vitro Ames Salmonella typhimurium assay did not show any increase in mutagenicity of smoke condensate from test cigarettes designed for studies 1 and 2 as compared to the reference. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure. Exposure to smoke from reference or test cigarettes in both studies induced increases in blood carboxyhemoglobin ((COHb)) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period. Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes.     

CYTOTOXICITY ASSESSMENT OF WHOLE SMOKE AND VAPOR PHASE OF MAINSTREAM AND SIDESTREAM CIGARETTE SMOKE FROM THREE KENTUCKY REFERENCE CIGARETTES

Assessment of the cytotoxicity of mainstream and sidestream cigarette sm oke has traditionally involved exposure of cell cultures to the particulate m atter of smoke. For a m ore com plete assessment of cigarette sm oke cytotoxicity, a technique (cellular smoke exposure technique or CSET) was developed to directly expose mammalian cell cultures to either whole mainstream or sidestream cigarette sm oke. The objective of this study was to com pare the cytotoxicity of whole smoke or vapor phase from mainstream or sidestream sm oke of three Kentucky reference cigarettes. The cigarettes com pared were a high ''tar''cigarette (2R1), a low ''tar'' cigarette (1R4F), and an ultra low ''tar'' cigarette (1R5F). Cytotoxicity was assessed in two cell types (WB rat liver cells and CHO cells) using the neutral red cytotoxicity assay. The order of cytotoxicity of m ainstream smoke from the three cigarettes expressed on a per cigarette basis was 2R1 > 1R4F > 1R5F. Sidestream sm oke from all three reference cigarettes was more toxic than the respective mainstream smoke on a per cigarette basis. The vapor phase of mainstream or sidestream smoke was the major contributor to the cytotoxicity of the whole cigarette smoke. Finally, the com parative trends in cytotoxicity between the smoke from the three reference cigarettes was similar in the two cell types, but CHO cells were more sensitive. CSET is a useful system to assess the cytotoxicity of cigarette smoke and m ay serve as an appropriate adjunct to the use of isolated particulate matter for the in vitro toxicological assessment of cigarette smoke and other aerosols.     

Effects of Flavoring and Casing Ingredients on the Toxicity of Mainstream Cigarette Smoke in Rats

A series of in vitro and in vivo studies evaluated the potential effects of tobacco flavoring and casing ingredients. Study 1 utilized as a reference control cigarette a typical commercial tobacco blend without flavoring ingredients, and a test cigarette containing a mixture of 165 low-use flavoring ingredients. Study 2 utilized the same reference control cigarette as used in study 1 and a test cigarette containing eight high-use ingredients. The in vitro Ames Salmonella typhimurium assay did not show any increase in mutagenicity of smoke condensate from test cigarettes designed for studies 1 and 2 as compared to the reference. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure. Exposure to smoke from reference or test cigarettes in both studies induced increases in blood carboxyhemoglobin ((COHb)) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period. Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference 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 microg total particulate matter/l for 90 days, 6h/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.     

Discriminatory power of standard toxicity assays used to evaluate ingredients added to cigarettes

A tiered approach for testing ingredients in a cigarette matrix was developed and includes chemical-analytical testing and a standard battery of biological toxicity assays. These assays were adapted for comparative evaluation of mainstream smoke from experimental cigarettes with or without ingredients at various inclusion levels. This adaptation to test cigarette mainstream smoke may impact assay response. Since it is difficult to a priori determine discriminatory power, it was evaluated using a large experimental dataset from a multi-year program of cigarette ingredient testing performed at two separate laboratories. A statistical method, minimum detectable difference (MDD), was used as a measure of assay discriminatory power. MDD of cigarette smoke constituents ranged from 6% to 29% of the average. Salmonella mutagenicity and cytotoxicity test MDDs ranged from 20% to 81% and 18% to 49%, respectively. Body weight gain in 90-day nose-only inhalation studies yielded an MDD of 30-40%. Histopathological findings with severity scores between 0.5 and 1.5 had the lowest MDDs of 23% and higher. In general, discriminatory power decreased with increasing biological complexity and toxicological relevance of the assay. Beyond statistical analysis, however, a weight-of-the-evidence analysis by experienced researchers is required for toxicological assessment of a cigarette ingredient.     

Cytotoxicity and gene expression profiles in cell cultures exposed to whole smoke from three types of cigarettes.

The purpose of this study was to evaluate and compare the cytotoxicity and gene expression profiles in cell cultures exposed to whole smoke generated from a full flavor cigarette (Test 1), a low tar cigarette (Test 2), and an ultra-low tar cigarette (Test 3). In addition, a reference cigarette 2R4F was evaluated for cytotoxicity. Neutral red (NR) cytotoxicity assay was performed to determine relative cell death at each exposure concentration (n = 6). LC(50) was generated using wet total particular matter (WTPM), cigarette number, or nicotine concentrations. The overall order of cytotoxicity was Test 1 > 2R4F approximately Test 2 > Test 3. Cell culture samples were collected for RNA extraction at WTPM concentrations of each cigarette that gave similar nicotine concentrations. Affymetrix mouse whole genome 430 2.0 array was used to characterize the gene expression profiles for each cigarette. A total of 598 genes in Test 1, 176 genes in Test 2, and 234 genes in Test 3 samples were differentially expressed compared to the concurrent sham controls. The major biological processes associated with the changed genes in Test 1 samples were down-regulated DNA replication and cell proliferation; the same biological processes were much less affected in Test 2 and Test 3 samples. The common findings in all three cigarettes types were increased glutathione biosynthesis/consumption and inflammatory response, which are known biological effects caused by smoke exposure. The most significantly up-regulated genes were CYP1A1, GSTs, Hmox1, and Procr in smoke-exposed samples, which are either related to well-studied mechanisms of smoke exposure-related diseases or potential new biomarkers for assessing and monitoring biological effects of cigarette smoke exposure in vivo and in smokers. In summary, both the NR cytotoxicity assay and gene expression profiling were able to differentiate the three types of test cigarettes, and the results demonstrated reduced biological effects for the Test 2 and Test 3 cigarettes compared to the Test 1 cigarette in BALB/c-3T3 Cells.     

In vitro bioactivity of combustion products from 12 tobacco constituents.

Twelve chemical components of tobacco leaf, representing 50% of its dry weight, were individually combusted and the bioactivities of their combustion products i.e. total particulate matter (TPM) were assayed using three in vitro tests. These components included carbohydrates, amino acids, proteins, polyphenols and carboxylic acids. The mutagenic potencies were assessed with the Salmonella mutagenicity assay (S. typhimurium TA98 and TA100). The induction of chromosomal damage, determined with the micronucleus test (IVMNT), and the neutral red uptake cytotoxicity test (NRU), were conducted on V79 hamster lung fibroblast cells. The Salmonella mutagenicity test and IVMNT were conducted with and without rat liver microsomal S9 fraction. Salmonella mutagenicity data confirmed the mutagenicity of TPM samples obtained from nitrogenous compounds (amino acids and proteins). The IVMNT showed that precursors of phenols in smoke (i.e. polyphenols) exhibited significantly higher levels of toxicity compared to other tobacco components. While S9 activation amplified the Salmonella mutagenicity response to combustion products, it significantly inhibited the toxicity measured with the IVMNT. NRU data demonstrated the increasing cytotoxicity induced following longer exposure time to TPM samples from nitrogenous and phenolic components. This study is the first to characterize the toxicity of the combustion products of major tobacco constituents. Our data suggest different mechanisms of toxicity and underline the relevance of using various bioassays.     

Toxicological comparisons of three styles of a commercial U.S. cigarette (Marlboro with the 1R4F reference cigarette

Toxicological comparisons were made of three commercial cigarettes, namely Marlboro full flavor, Marlboro Lights, and Marlboro Ultra Lights, with the 1R4F reference cigarette. The main comparison was a 90-d inhalation study with mainstream smoke at 150 mg total particulate matter per cubic meter, in Sprague-Dawley rats using 6 h/d and 7 d/w exposures. The principal endpoint was histopathology of the respiratory tract, along with examinations of free lung cell counts after broncho-alveolar lavage. Additional studies on mainstream smoke included Salmonella mutagenicity, cytotoxicity of particulate and gas/vapor phases, and analytical chemistry. The exposures produced effectively the same responses in each of the four groups, and the histopathology results in the commercial cigarette groups were also effectively the same. The 1R4F was also tested at 75 and 200 mg/m(3), and most of the histopathology results obtained here showed dose-response relationships. The free lung cell responses were similar in the 1R4F/commercial cigarette comparison, and there were dose-related changes in the 1R4F groups, most notably for neutrophils. Most of the changes produced in the 90-d of exposure were resolved in a 42-d post-inhalation period. Responses in the in vitro and analytical assays for the four cigarettes were in general similar, when data were expressed either per mg TPM or per mg tar yield. There were judged to be no toxicologically meaningful differences between the profiles evaluated at similar smoke concentrations for the three commercial cigarettes and for the 1R4F using these assays.     

Chemical composition, cytotoxicity and mutagenicity of smoke from US commercial and reference cigarettes smoked under two sets of machine smoking conditions

Eight blended US market cigarettes, two blended reference cigarettes, one Bright tobacco only reference cigarette and an electrically heated prototype cigarette (EHC) were smoked under US Federal Trade Commission (FTC)/International Organisation for Standardisation (ISO) conditions and under Massachusetts Department of Public Health (MDPH) conditions. Smoke was analysed for chemical composition and in vitro toxicity. Yields (quantity/cigarette) of smoke constituents were higher under MDPH conditions compared to FTC/ISO conditions (market and reference average approximately 2.5 times; EHC approximately 1.6 times). Consistent with the higher yields, in vitro toxicity per cigarette was also higher under MDPH conditions. Concentrations (quantity/mg TPM) of nearly all smoke constituents measured decreased with increasing total particulate matter (TPM) yields as regression analyses indicated. Higher TPM yields also tended to be associated with slightly less cytotoxic and mutagenic activity per milligram TPM. Blended reference cigarettes tracked market cigarettes with similar TPM yield. The Bright cigarette displayed high cytotoxicity but low mutagenicity, while in vitro activity of the EHC was remarkably low. The TPM-dependent decreases for the market range of 5-20 mg TPM/cigarette were about 20%, irrespective of whether the increased yields were due to smoking conditions or cigarette construction. At the same TPM yield, the smoke constituent concentrations and in vitro toxicity were similar for low- and high-yield cigarettes.     

Comparison of the cytotoxic and mutagenic potential of liquid smoke food flavourings, cigarette smoke condensate and wood smoke condensate.

Although products of pyrolysis are often cytotoxic and mutagenic, the relationship between the type of material pyrolysed and the toxicity of the resulting pyrolysis products is poorly understood. The objective of this study was to evaluate and compare the cytotoxicity and mutagenicity of several types of common pyrolysis products. The cytotoxicity and mutagenicity of these products were assessed by using neutral red uptake and Ames mutagenicity assays, respectively. The biological activities of four liquid smoke food flavourings (LSF) were compared with two other pyrolysis-derived materials; cigarette smoke condensate (CSC) and a wood smoke condensate (WSC). Results indicated all of the mixtures exhibited a concentration-dependent cytotoxic response. The CSC and WSC were less cytotoxic than three of the LSFs, but more cytotoxic than one of the brands. The CSC was mutagenic in two Salmonella strains; however, none of the LSFs or WSC was mutagenic using TA98, and only three of the LSFs were positive with TA100. The six pyrolysis-derived materials evaluated in this study showed differing patterns and magnitudes of cytotoxicity and mutagenicity. These results indicate that the cytotoxicity and mutagenicity of complex mixtures derived from pyrolysis products are affected by the type of material pyrolysed and/or the method used to prepare the mixture. The cytotoxic potential of some commercial smoke flavourings is greater than cigarette smoke condensate and several of the food flavourings are mutagenic in one Salmonella strain.