Toxicological evaluation of an electrically heated cigarette. Part 4: Subchronic inhalation toxicology

The biological activity of mainstream smoke from an electrically heated cigarette (EHC) with controlled combustion and from the University of Kentucky Reference Cigarette 1R4F was determined in Sprague Dawley rats exposed nose-only for 90 days, 6 h a day, 7 days per week. For an equivalent response comparison between the two cigarette types, two doses were chosen for the EHC where the anticipated results were in the dynamic range of the 1R4F dose-response curve (four concentrations) for most end points. The number of cigarettes smoked per m(3) of diluted smoke resulted in total particulate matter concentrations of 40 and 90 microg l (-1) for the EHC and 40-170 microg l (-1) for the 1R4F. Biomonitoring indicated achievement of target doses. Mainstream smoke yields were lower for the EHC, with the exception of formaldehyde. No smoke-related mortality, remarkable in-life observations or abnormal gross pathological findings were observed. Smoke- and dose-related clinical pathology and organ weight changes included: increases in segmented neutrophils, some liver parameters and lung and adrenal weight relative to body weight; and decreases in lymphocytes, glucose concentration and spleen weight. Smoke-related histopathological findings in the respiratory tract included epithelial cell hyperplasia, squamous metaplasia, atrophy and accumulation of pigmented alveolar macrophages; they were mostly dose-dependent, more pronounced in the upper than lower respiratory tract and completely or partially reversed by 6 weeks post-inhalation. Qualitatively, the biological effects seen for the EHC and the 1R4F were comparable and similar to those observed in other mainstream smoke inhalation studies. Quantitatively, the biological activity of the EHC mainstream smoke was, on average, 65% lower than that of the 1R4F mainstream smoke on an equal cigarette basis and equivalent activity on an equal TPM basis.     

Toxicological evaluation of an electrically heated cigarette. Part 2: Chemical composition of mainstream smoke

The chemical composition of mainstream smoke from an electrically heated cigarette (EHC) and that of mainstream smoke from the University of Kentucky Reference Cigarette 1R4F was analyzed.In contrast to the 1R4F, which is a conventional, lit-end cigarette, the EHC is smoked in a microprocessor-controlled lighter with electrical heater elements. The electrical heating causes the tobacco under the heater element to burn at a low temperature during each puff. A comprehensive list of chemical constituents was analyzed in mainstream smoke. The list is a combination of those compounds suggested for analysis in cigarette smoke by a US Consumer Product Safety Commission proposal in 1993, and those cigarette smoke constituents identified by the International Agency on Research on Cancer as being present in cigarette smoke and characterized as carcinogens. The low pyrolysis/combustion temperature of tobacco in the EHC causes distinct shifts in the composition of the smoke compared with a conventional cigarette. A significant drop was seen in the yields of almost all toxicologically relevant constituents. On a per cigarette basis almost two-thirds of the constituents were reduced by at least 80%, whereas on an equal total particulate matter basis about two-thirds of the constituents were reduced by at least 50%, with many constituents reduced by more than 90%.     

Effect of cigarette filters on the chemical composition and in vitro biological activity of cigarette mainstream smoke

The objective of this study was to evaluate the effects of cigarette filters on the chemical composition and toxicity of cigarette mainstream smoke. In this work, we used three types of cigarettes, including non-filter 2R4F cigarettes, cellulose acetate (CA)-filter 2R4F cigarettes, and carbon dual-filter 2R4F cigarettes. The cytotoxicity of TPM obtained from the filter cigarettes was not different from that of the non-filter cigarettes on an equal TPM basis. However, the EC₅₀ vlaue of GVP from carbon-filter cigarettes were 40.9 puffs/L, thereby indicating the cytotoxicity of these cigarettes was approximately 37% and 21% lower than non-filter and CA-filter cigarettes, respectively. The cytotoxicity of GVP was correlated with carbonyl components. The mutagenicity of TPM obtained from non-filter cigarettes, calculated on an equal TPM basis, was up to 30–40% lower than that of the filter cigarettes. When calculated on a per cigarette basis, the mutagenicity of CA or carbon-filter cigarettes was found to be 35% lower than that of the non-filter cigarettes. The results of chemical composition analyses revealed that the observed increase in aromatic amine compound yields on an equal TPM basis in filter cigarettes may be related with the mutagenic activity determined in Ames 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.     

Reduced toxicological activity of cigarette smoke by the addition of ammonium magnesium phosphate to the paper of an electrically heated cigarette: smoke chemistry and in vitro cytotoxicity and genotoxicity

The effects of the addition of ammonium magnesium phosphate (AMP) to the paper of an electrically heated cigarette (EHC) prototype on smoke composition and toxicity were quantified and the underlying mechanisms investigated. Smoke from EHC prototypes with and without AMP and from conventional cigarettes, i.e. the University of Kentucky Standard Reference Cigarette 1R4F and eight American-blend market cigarettes, was compared. Endpoints for comparison were smoke chemistry, where toxic constituents were measured, cytotoxic activity, as measured in murine fibroblasts embryo cells by the Neutral Red Uptake Assay, and genotoxic activity, as measured in bacteria by the Salmonella Reverse Mutation Assay and in murine lymphoma cells by the TK Assay. The addition of AMP to the EHC led to a reduction of toxic substances and toxicological activity of approximately 30% compared to the EHC without AMP. Compared to the conventional cigarettes, the EHC with AMP showed reductions of 75-90%. Smoke from the EHCs generated in nitrogen atmospheres supplemented with different concentrations of ammonia and oxygen was assayed for its in vitro cytotoxicity and genotoxicity. The results indicate that the ammonia released by AMP at the heating site of the EHC is responsible for the reductions in cytotoxicity and mutagenicity for the EHC with AMP compared with the EHC without AMP. Thus, while the EHC approach distinctly reduces toxic smoke constituents compared to conventional cigarettes, the use of AMP in the paper of an EHC leads to further distinct reductions. In the study presented here, in vitro assays were used as quantitative tools to investigate toxicity-related mechanisms.     

Effect of smoking conditions and methods of collection on the mutagenicity and cytotoxicity of cigarette mainstream smoke.

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

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.     

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.     

Reduced toxicological activity of cigarette smoke by the addition of ammonia magnesium phosphate to the paper of an electrically heated cigarette: subchronic inhalation toxicology

Cigarette smoke is a complex chemical mixture that causes a variety of diseases, such as lung cancer. With the electrically heated cigarette smoking system (EHCSS), temperatures are applied to the tobacco below those found in conventional cigarettes, resulting in less combustion, reduced yields of some smoke constituents, and decreased activity in some standard toxicological tests. The first generation of electrically heated cigarettes (EHC) also resulted in increased formaldehyde yields; therefore, a second generation of EHC was developed with ammonium magnesium phosphate (AMP) in the cigarette paper in part to address this increase. The toxicological activity of mainstream smoke from these two generations of EHC and of a conventional reference cigarette was investigated in two studies in rats: a standard 90-day inhalation toxicity study and a 35-day inhalation study focusing on lung inflammation. Many of the typical smoke exposure-related changes were found to be less pronounced after exposure to smoke from the second-generation EHC with AMP than to smoke from the first-generation EHC or the conventional reference cigarette, when compared on a particulate matter or nicotine basis. Differences between the EHC without AMP and the conventional reference cigarette were not as prominent. Overall, AMP incorporated in the EHC cigarette paper reduced the inhalation toxicity of the EHCSS more than expected based on the observed reduction in aldehyde yields.     

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.     

Reduced Toxicological Activity of Cigarette Smoke by the Addition of Ammonia Magnesium Phosphate to the Paper of an Electrically Heated Cigarette: Subchronic Inhalation Toxicology

Cigarette smoke is a complex chemical mixture that causes a variety of diseases, such as lung cancer. With the electrically heated cigarette smoking system (EHCSS), temperatures are applied to the tobacco below those found in conventional cigarettes, resulting in less combustion, reduced yields of some smoke constituents, and decreased activity in some standard toxicological tests. The first generation of electrically heated cigarettes (EHC) also resulted in increased formaldehyde yields; therefore, a second generation of EHC was developed with ammonium magnesium phosphate (AMP) in the cigarette paper in part to address this increase. The toxicological activity of mainstream smoke from these two generations of EHC and of a conventional reference cigarette was investigated in two studies in rats: a standard 90-day inhalation toxicity study and a 35-day inhalation study focusing on lung inflammation. Many of the typical smoke exposure-related changes were found to be less pronounced after exposure to smoke from the second-generation EHC with AMP than to smoke from the first-generation EHC or the conventional reference cigarette, when compared on a particulate matter or nicotine basis. Differences between the EHC without AMP and the conventional reference cigarette were not as prominent. Overall, AMP incorporated in the EHC cigarette paper reduced the inhalation toxicity of the EHCSS more than expected based on the observed reduction in aldehyde yields.     

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.     

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.     

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.     

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.     

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/m³, 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.     

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.     

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.     

In utero exposure to 1R4F reference cigarette smoke: evaluation of developmental toxicity.

The potential developmental effects of 1R4F reference cigarette smoke were examined using Sprague-Dawley rats exposed for 2 h/day, 7 days/week, by nose-only inhalation at target mainstream smoke concentrations of 150, 300, and 600 mg/m3 total particulate matter (TPM). Males were exposed 4 weeks prior to and during mating, with females exposed 2 weeks prior to mating and during mating, and through gestation day (GD) 20. Sham controls received filtered air to simulate nose-only exposure, while cage controls were maintained untreated. Smoke exposure was confirmed through biomarker evaluation (parental: carboxyhemoglobin, nicotine, and cotinine; fetal: nicotine and cotinine). Characteristic cigarette smoke-related histopathologic changes including nasal epithelial hyperplasia and squamous metaplasia and pigmented macrophages in the lung were observed in all exposed parental groups. Maternal toxicity during gestation was indicated at smoke concentrations of 300 and 600 mg TPM/m3, where corrected total body weight gain was significantly (p 相似文献