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-microg 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.     

Chemical and biological studies of a cigarette that heats rather than burns tobacco

Cigarettes can be developed that heat rather than burn tobacco. Such products would be expected to have less "tar" and other combustion products than cigarettes that burn tobacco. With one product of this type, benzo(a)pyrene, N-nitrosamines, phenolic compounds, acetaldehyde, acrolein, hydrogen cyanide, and N-heterocyclic compounds have been reduced 10- to 100-fold compared to the Kentucky reference (1R4F) cigarette, a representative low-tar cigarette. The yields of nicotine and carbon monoxide from this new cigarette are less than the yields of 95% and 75%, respectively, of the cigarettes sold in the United States during 1988. Nicotine absorption from smoking this new cigarette is not significantly different from that of tobacco-burning cigarettes yielding equivalent levels of nicotine. The urine mutagenicity of smokers of new cigarettes is significantly less (P less than .05) than that of smokers of tobacco-burning cigarettes and is not significantly different (P greater than .10) from that of nonsmokers. We conclude that cigarettes which heat rather than burn tobacco can reduce the yield of tobacco combustion products. This simplification of smoke chemistry had no effect on nicotine absorption in smokers and resulted in a reduction of biological activity in smokers as measured by urine mutagenicity.     

Toxicological analysis of low-nicotine and nicotine-free cigarettes

Low-nicotine and nicotine-free cigarettes are commercially available under the brand-name Quest. Some consumers may believe that these are safer cigarettes, and they may smoke more cigarettes or inhale more smoke to compensate for low nicotine yields. Thus, we have studied the toxicological effects of these two cigarettes and compared them with the Kentucky reference cigarette 2R4F. Also, the availability of nicotine-free cigarettes allows for the assessing the role of nicotine in cigarette smoke. In addition to nicotine, some tobacco-specific nitrosamines, aldehydes, and volatile organic compounds were also reduced in the Quest cigarettes compared to the 2R4F. However, aromatic amines were higher in the nicotine-free compared with low nicotine cigarettes. The Ames test revealed that cigarette smoke condensates from the nicotine-free (CSC-F), low nicotine (CSC-L) and 2R4F (CSC-R) cigarettes had a similar mutagenic potency. Exposure to any CSC caused a similar dose-dependent LDH leakage from normal human bronchial epithelial cells. However, CSC-F had more inhibitory effects on the cell growth than CSC-L and CSC-R. Adding nicotine to the CSC-F attenuated this inhibition. Both Quest CSCs decreased gap junction intercellular communication and caused cell cycle arrest. CSC exposure increased cytoplasmic nucleosomes, sub-G1/G0 population and apoptotic comet tails. Proapoptotic protein Bax increased independent of p53 induction after exposure to CSC-F. In conclusion, these studies are not consistent with a perception that low-nicotine or nicotine-free cigarettes may have less toxicity in human cells. Nicotine, as it exists in CSC, attenuates cytotoxicity possibly in part through inhibition of apoptotic pathways.     

Gap junction intercellular communication and cytotoxicity in normal human cells after exposure to smoke condensates from cigarettes that burn or primarily heat tobacco.

Heating tobacco, rather than burning it, reduces tobacco combustion and pyrolysis products. This study tested the hypothesis that the simplified smoke chemistry of a cigarette which primarily heats tobacco (TOB-HT) significantly reduces the potential to alter the structure or function of cellular plasma membranes relative to low "tar" 1R4F and ultra low "tar" lR5F Kentucky reference cigarettes which burn tobacco. Gap junction intercellular communication (GJIC) and lactate dehydrogenase release (LDH) were used to quantify functional and structural changes to the plasma membrane, respectively. Cigarette smoke condensate (CSC) from the mainstream smoke of TOB-HT, lR4F and 1R5F cigarettes were compared in the GJIC and LDH release assays following a 1-hr exposure in vitro. Human bronchial/tracheal epithelial cells, coronary artery endothelial cells, coronary artery smooth muscle cells, foreskin keratinocytes and the WB-344 rat liver epithelial cell line were studied. TOB-HT did not inhibit GJIC in any of the human cell types tested (P0.05) at concentrations where 1R4F and lR5F did inhibit GJIC (P<0.05). TOB-HT did not elevate LDH release (P0.05) when tested at concentrations where lR4F and lR5F did elevate LDH release (P<0.05). Our results suggest that CSC from TOB-HT cigarettes is less damaging to the structure or function of the cellular plasma membranes of a variety of human cell lines than CSC from 1R4F and 1R5F tobacco burning reference cigarettes.     

A quantitative approach to assessing intercellular communication: studies on cigarette smoke condensates

Analyses of intercellular communication is useful for assessing the effects of chemical treatment on the function of mammalian cell membranes in vitro. The objective of this study was to quantify and compare the activity of mainstream cigarette smoke condensate (CSC) from tobacco-heating and tobacco-burning cigarettes on both the rate and total amount of intercellular communication in vitro. Lucifer yellow uptake and lactate dehydrogenase release assays were used to evaluate plasma membrane toxicity. Gap junction intercellular communication (GJIC) was determined by quantifying fluorescence redistribution after photobleaching (FRAP) following a 1-hr exposure to concentrations of CSCs which were not toxic to the plasma membrane. GJIC was quantified in rat hepatic epithelial cells (WB cells) and human skin fibroblasts (MSU-2 cells) synchronized in the G1 phase of the cell cycle. In each of the cell types tested, CSC from tobacco-heating cigarettes did not inhibit GJIC at concentrations, where CSC from tobacco-burning cigarettes significantly inhibited both the total amount and the rate of GJIC. These results indicate that mainstream smoke condensate of cigarettes which heat tobacco is less biologically active than mainstream smoke condensate of cigarettes that burn tobacco as determined by in vitro gap junction intercellular communication.     

Comparative study of smoke condensates from 1R4F cigarettes that burn tobacco versus ECLIPSE cigarettes that primarily heat tobacco in the SENCAR mouse dermal tumor promotion assay.

Numerous chemical and toxicological studies indicate that smoke from ECLIPSE, a cigarette that primarily heats rather than burns tobacco, is simplified and reduced in specific chemicals believed to be associated with smoking-related diseases, and demonstrates reduced smoke toxicity and biological activity in vitro when compared to conventional tobacco burning cigarettes. These data led to the hypothesis that cigarette smoke condensate (CSC) from ECLIPSE should have lower tumorigenicity than 1R4F condensate in the SENCAR mouse dermal tumor promotion assay. Female SENCAR mice were initiated with a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with ECLIPSE or 1R4F CSC. Dermal application of 10, 20, or 40 mg ECLIPSE or 1R4F CSC three times/week for 29 weeks did not alter body weights, survival or other indicators of subchronic toxicity. In DMBA-initiated mice, there were significant increases in both the number of microscopically confirmed tumor-bearing animals and total number of microscopically confirmed dermal tumors at all 1R4F CSC doses and the high-dose ECLIPSE CSC. However, the number of ECLIPSE tumor-bearing animals were reduced 83%, 93% and 67% at the low-, mid- and high-doses, respectively, compared to the 1R4F. Similarly, the total number of dermal tumors was reduced 91%, 94% and 87% at the low-, mid- and high-dose, respectively, compared to the 1R4F CSC. ECLIPSE CSC demonstrated dramatic reductions in dermal tumor promotion potential compared to 1R4F CSC.     

A comparison of the mainstream smoke chemistry and mutagenicity of a representative sample of the US cigarette market with two Kentucky reference cigarettes (K1R4F and K1R5F).

The incorporation of technologies into cigarettes such as filters, filter ventilation, porous cigarette papers, expanded tobacco and reconstituted tobacco sheet has resulted in cigarettes with a wide range of "tar" yields. The objectives of this study were to characterize the US cigarette market according to "tar" category (i.e. full flavor, FF; full flavor low tar, FFLT; or ultra low tar, ULT) and to determine whether the Kentucky reference cigarettes K1R4F and K1R5F are representative of FFLT and ULT cigarettes, respectively. As a means of characterization and comparison, the mainstream smoke from a representative sample of commercially available cigarettes from each market segment and the K1R4F and K1R5F Kentucky reference cigarettes was analyzed for the presence and level of 18 selected chemical constituents. In addition, a measure of the mutagenic activity of the mainstream smoke condensate from these cigarettes was determined using an Ames Salmonella mutagenicity assay. All cigarettes were smoked according to US Federal Trade Commission (FTC) guidelines. Results indicated that, overall, mainstream smoke constituent levels are well predicted by FTC "tar" yield--constituent levels increased as "tar" delivery increased. Based on the selected analytes measured in mainstream smoke, the K1R4F reference cigarette was generally representative of the FFLT segment of the US cigarette market. The K1R5F reference cigarette was representative of the ULT segment of the US cigarette market for cigarettes with "tar" deliveries approximate to it. In terms of mutagenic activity, a direct relationship was also demonstrated on a per cigarette basis-revertants per cigarette increased with increasing "tar" delivery. There was a weak tendency (R-square = 0.12, P = 0.08) for specific activity (revertants/mg "tar") to increase with decreasing "tar" yield-lower "tar" products had a slightly higher specific activity. No significant differences (P > 0.05) were observed when the specific activities of the condensates from the K1R4F and K1R5F reference cigarettes were compared to the market segments that they were designed to represent, FFLT and ULT, respectively. Overall, these results support the use of the K1R4F and the K1R5F as acceptable reference cigarettes for comparative mutagenicity and smoke chemistry studies of cigarettes available on the US market.     

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.     

Development of a commercial cigarette “market map” comparison methodology for evaluating new or non-conventional cigarettes

A “market map” comparison methodology for cigarette smoke chemistry yields is presented. Federal Trade Commission machine-method smoke chemistry was determined for a range of filtered cigarettes from the US marketplace. These data were used to develop illustrative market maps for each smoke constituent as analytical tools for comparing new or non-conventional cigarettes to a sampling of the broader range of marketplace cigarettes. Each market map contained best-estimate “market-means,” showing the relationship between commercial cigarette constituent and tar yields, and yield “market ranges” defined by prediction intervals. These market map means and ranges are the basis for comparing new cigarette smoke yields to those of conventional cigarettes. The potential utility of market maps for evaluating differences in smoke chemistry was demonstrated with 1R4F and 2R4F Kentucky reference cigarettes, an Accord™ cigarette, and an Advance™ cigarette. Conventional cigarette tobacco nicotine, nitrate, soluble ammonia, and tobacco specific nitrosamine levels are reported. Differences among conventional cigarette constituent yields at similar tar levels were explained in part by the chemical composition range of those cigarette tobaccos. The study also included a comparison of smoke constituent yields and in vitro smoke cytotoxicity and mutagenicity assay results for the 1R4F Kentucky reference cigarette and its replacement 2R4F. Significant smoke yield differences were noted for lead, NNK, and NNN. The majority of their smoke constituent yields were within the market range developed from the sampled conventional cigarettes. Within the sensitivity and specificity of the in vitro bioassays used, smoke toxic activity differences for the two reference cigarettes were not statistically significant. These results add to the limited information available for the 2R4F reference cigarette.     

Development of a commercial cigarette "market map" comparison methodology for evaluating new or non-conventional cigarettes

A "market map" comparison methodology for cigarette smoke chemistry yields is presented. Federal Trade Commission machine-method smoke chemistry was determined for a range of filtered cigarettes from the US marketplace. These data were used to develop illustrative market maps for each smoke constituent as analytical tools for comparing new or non-conventional cigarettes to a sampling of the broader range of marketplace cigarettes. Each market map contained best-estimate "market-means," showing the relationship between commercial cigarette constituent and tar yields, and yield "market ranges" defined by prediction intervals. These market map means and ranges are the basis for comparing new cigarette smoke yields to those of conventional cigarettes. The potential utility of market maps for evaluating differences in smoke chemistry was demonstrated with 1R4F and 2R4F Kentucky reference cigarettes, an Accord cigarette, and an Advance cigarette. Conventional cigarette tobacco nicotine, nitrate, soluble ammonia, and tobacco specific nitrosamine levels are reported. Differences among conventional cigarette constituent yields at similar tar levels were explained in part by the chemical composition range of those cigarette tobaccos. The study also included a comparison of smoke constituent yields and in vitro smoke cytotoxicity and mutagenicity assay results for the 1R4F Kentucky reference cigarette and its replacement 2R4F. Significant smoke yield differences were noted for lead, NNK, and NNN. The majority of their smoke constituent yields were within the market range developed from the sampled conventional cigarettes. Within the sensitivity and specificity of the in vitro bioassays used, smoke toxic activity differences for the two reference cigarettes were not statistically significant. These results add to the limited information available for the 2R4F reference cigarette.     

Subchronic inhalation by rats of mainstream smoke from a cigarette that primarily heats tobacco compared to a cigarette that burns tobacco

A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smoke-exposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.     

SUBCHRONIC INHALATION BY RATS OF MAINSTREAM SMOKE FROM A CIGARETTE THAT PRIMARILY HEATS TOBACCO COMPARED TO A CIGARETTE THAT BURNS TOBACCO

A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smokeexposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.     

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.     

Cytotoxicity of eight cigarette smoke condensates in three test systems: Comparisons between assays and condensates

Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose–response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells.     

Cytotoxicity of eight cigarette smoke condensates in three test systems: Comparisons between assays and condensates

Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose–response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells.     

Cytotoxicity,mutagenicity, and tumorigenicity of mainstream smoke from three reference cigarettes machine-smoked to the same yields of total particulate matter per cigarette

The particle phase of mainstream smoke from three types of cigarettes was investigated in vitro in the Neutral Red cytotoxicity assay and the Salmonella typhimurium Reverse Mutation Assay (Ames Assay) and in vivo in the two-stage dermal tumorigenicity assay (Skin Painting Assay) in SENCAR mice. The cigarettes used were the Reference Cigarettes 1R5F, 2R4F, and 2R1F from the University of Kentucky, USA, which, when smoked according to the smoking regimen defined by the International Standards Organization (ISO), produce a yield of approximately 2, 12, and 26 mg total particulate matter (TPM)/cigarette, respectively. All cigarettes were machine smoked according to ISO and then again in such a way that the TPM yields per cigarette equaled the ISO TPM yields of the other two cigarette types.     

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.     

A review of in vitro methods to assess the biological activity of tobacco smoke with the aim of reducing the toxicity of smoke

In the last few years tobacco companies have been developing several research strategies in order to reduce the risks associated with smoking. These strategies include, for example, the refining of alternative cigarette designs that reduce the amount of hazardous chemicals in the mainstream smoke by introducing modified filters, and/or reducing the amount of biologically significant ingredients in tobacco-burning cigarettes. In the last few decades numerous studies have been published to assess the biological activity of tobacco smoke using in vivo and in vitro test systems. In this scenario a general scientific consensus on how to measure and characterize the risk associated with cigarette smoke is still lacking. Short-term in vitro assays, which are widely accepted by regulatory agencies around the world, are useful tools to evaluate both the biological activity and the progress towards a reduction of tobacco smoke toxicity. These assays could be mainly applied to evaluate cytotoxicity and genotoxicity properties on whole cigarette smoke as well as condensates or fractions of whole smoke. Cytotoxicity induction can be measured as cellular viability and growth rates using different end-points. Otherwise, the target of genotoxicity studies is the DNA molecule. For genotoxicity evaluation, the end-points and cell systems should be chosen from those that are relevant and appropriate as clinical surrogate markers. In this respect, the occurrence of early biological effect markers, such as mutational or clastogenic events (point mutations, frameshifts, micronuclei, SCE, DNA adducts) in bacterial and mammalian cells should be studied in a tiered approach following the guidelines of regulatory agencies. The choice of criteria shall be matter of discussion.     

A responsive, sensitive, and reproducible dermal tumor promotion assay for the comparative evaluation of cigarette smoke condensates

The mouse dermal initiation/promotion bioassay has been used for several decades to study cigarette smoke condensates (CSCs). However, these studies have used highly variable methodologies that differ in the manner of CSC collection, duration of treatment, mouse strain, number of mice and endpoints measured. In this report, a protocol that uses female SENCAR mice and standardizes many of the procedures is presented. A reference cigarette (University of Kentucky 1R4F), readily available to researchers, was used. This report presents the combined data from four independent studies. Female, SENCAR mice (40/group) were treated with a single dose (75microg) of dimethylbenz[a]anthracene (DMBA) as an initiator, followed 1 week later by treatment (three times/week) with 10, 20 or 40mg "tar"/application of 1R4F CSC for 29 weeks. There were no treatment-related effects on body weights. Histological diagnosis of all masses at study termination indicated a dose-dependent increase in the number of tumor-bearing mice and total tumor number. These studies support the conclusion that the 1R4F cigarette is suitable for use as a reference standard and the protocol presented is an appropriate and standardized model suitable for the comparative evaluation of CSC.     

Analysis of potential transfer of continuous glass filament from Eclipse cigarettes

This study was designed to determine if a prototype of the Eclipse cigarettes, which uses a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yielded CGFs via mainstream smoke. A method was developed that used electrostatic precipitation with a greater than 99% collection efficiency of mass to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition that was more than twice the Federal Trade Commission (FTC) standard. The cigarettes were subjected to handling procedures that simulated commercial shipping conditions prior to smoking. CGFs were intentionally added to a series of smoke condensate samples to determine CGF recovery efficiency. The recovery efficiency was determined for a series of four internal standards added to smoke condensate. The recovery efficiency was 86% for the Eclipse 5-014 prototype. The number of CGFs in smoke condensate collected from the Eclipse 5-014 prototype was approximately 0.06 +/- 0.02 CGFs per cigarette (+/- standard deviation), including the background counts of CGFs and 0.03 CGFs per cigarette, when corrected for background contributions. The number of CGFs found in smoke condensates for this prototype was not statistically distinguishable from zero or background in these experiments, which were capable of detecting transfer rates of greater than 0.2 CGFs per cigarette.