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

Toxicological Sciences, Sep 1997

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NOx, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.

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

D. W. Bombick 0 1 B. Reed Bombick 0 1 P. H. Ayres.t K. Putnam 0 1 J. Avalos 0 1 M. F. Borgerding 0 D. J. Doolittle 0 1 0 these assays. S 1997 Society of Toxicology 1 Environmental and Molecular Toxicology Division 2 Analytical Chemistry Division, R. J. Reynolds Tobacco Company , Winston-Salem, North Carolina 27102 Cigarette Description - A new cigarette prototype which contains a novel carbon filter and a low-nitrogen tobacco blend has been developed. The new cigarette (designated EXP-C) was in the "full-flavor low-tar" category and was compared with five other full-flavor low-"tar" cigarettes, including two prototypes and three commercially available cigarettes. A brief description and designation for each cigarette are given in Table 1. 0272-0590/97 $25.00 Copyright O 1997 by the Society of Toxicology. AJI rights of reproduction in any form reserved. All six cigarettes were similar in FTC (Federal Trade Commission) values for total paniculate matter, tar, CO, and nicotine content The carbon filter consists of a gathered web of paper incorporating a carbonaceous material. The paper is gathered so as to include a large number of longitudinal extended channels and the channels provide a cross-sectional void area of about 5 to 30% of the filter element (U.S. Patent No. 5,360,023). The filter is capable of removing condensable gas-phase components (including carbonyls such as formaldehyde, acetaldehyde, and acrolein) from mainstream tobacco smoke to a significant degree. The carbon filter is different from existing commercial carbon-filtered cigarettes because the carbon scrubber filter consists of a gathered web of paper with highly refined charcoal (40 /im particle average) incorporated into the paper matrix of the filter and does not consist of a cavity filter with charcoal granules or a rolled paper with surface-applied charcoal powder. Flue-cured tobacco containing less nitrogenous compounds (Wynder and Hoffmann, 1967) was used in the blending for the low-nitrogen cigarette tobaccos. These low-nitrogen tobaccos may reduce the amount of mutagenic aromatic amine compounds in the smoke (Hoffmann et ai, 1980; Schmeltz and Hoffmann, 1977). Chemical Analyses of Mainstream Cigarette Smoke The standard FTC puffing regimen (35-ml puff volume, 2-sec duration, once per minute) was used to generate mainstream smoke for chemical analyses (Fed. Reg., 1967). The FTC method was used to determine total paniculate matter (TPM), nicotine, and tar (Pillsbury et ai, 1969). Carbon monoxide was determined by nondispersive infrared spectroscopy after collecting the gas phase (Horton and Guerin, 1974). The following analyses were performed on whole smoke. Ammonia was determined by a colorimetric procedure (Harrell et ai, 1975). Acrylonitrile, benzene, isoprene, and 1,3-butadiene were determined by gas chromatography with mass selective detection with conditions adapted from a published procedure (Byrd et at., 1990). Formaldehyde, acetaldehyde, acetone, and acrolein were determined by liquid chromatography with fluorescence detection after derivatization with 2-diphenyl-acetyl-l,3-indandione-l-hydrazone to form azine derivatives (Borgerding et ai, 1984). Nitrogen oxides were determined by chemiluminescence (Neurath et ai, 1976). Hydrogen cyanide was determined by trapping the compound on ascarite followed by water extraction and colorimetric end determination (Collins et ai, 1970). The aggregate vapor-phase mass was calculated by totaling the determined quantities of 11 vapor-phase components (ammonia, acrylonitrile, acetone, acrolein, acetaldehyde, benzene, formaldehyde, NO,, HCN, 1,3butadiene, and isoprene) from the mainstream smoke of one cigarette. These 11 vapor-phase components were chosen because of their significant proportion of cigarette smoke vapor-phase components (Guerin et ai, 1992) and/ or their strong irritancy or cytotoxic potential. Collection of Cigarette Smoke Condensate (CSC) Cigarette smoke condensates from the six cigarettes were prepared by smoking the cigarettes under standard FTC conditions (35-ml puff volume, 2-sec duration, once per minute) on a smoking machine. The cigarette smoke condensates were collected on Cambridge filter pads and weighed. The filter pads were extracted with dimethyl sulfoxide (DMSO) to a final concentration of 10 mg tar per milliliter of DMSO based on the weight of material on the Cambridge filter pad (Pillsbury et ai, 1969). DMSO was used as the solvent control in all assays using CSC. Cellular Smoke Exposure Technology (CSET) CSET technology has been utilized to characterize reference cigarettes (Bombick et ai. in press) and is briefly described as follows: Mainstream cigarette smoke was generated by smoking the six cigarettes under FTC conditions on a 30-port rotating carousel as described by Baumgartner and Coggins (1980). Mainstream smoke from the smoke generator was diluted with humidified, HEPA (high-efficiency paniculate accumulation), and charcoal-filtered air and distributed to a two-tier exposure chamber constructed of PVC pipe as described by Cannon et ai (1983). Concentration in the chamber was controlled by a feedback-loop exposure control system as described by Ayres et ai (1990) and was verified by gravimetric determination of total paniculate matter. Several aluminum blocks wrapped with heating tape are designed to hold tissue culture flasks on a rocking platform. The temperature of the culture flasks was maintained at 37C by a programmable temperature regulator. Individual culture flasks were connected to the exposure chamber ports by food-grade silicone tubing and cells within the flask were exposed to mainstream cigarette smoke by drawing smoke from the smoke generator through the flask at a flow rate of 275 ml/min. Negative controls utilized humidified, HEPA filtered air drawn through the culture flask at a flow rate of 275 ml/min. The rocking platform allows the cells to oscillate between exposure to mainstream cigarette smoke and culture medium at an approximate rate of seven cycles per minute. Ames Bacterial Mutagenicity Assay Mutagenicity was assessed in the SalmonellaJmicrosome assay (Maron and Ames, 1983) with the preincubation modification described by Yahagi et ai (1975). A standardized liver homogenate (S9) was purchased from MolTox (Annapolis, MD). The S9 concentration in the S9 mix was 5% (v/v), and 0.5 ml of the S9 mix was added per plate. The CSC test sample in DMSO (not to exceed 50 fA per plate) was added to the S9 mix containing the test bacteria in a glass test tube maintained at 37C. The mixture was shaken and allowed to incubate for 20 min at 37C prior to the addition of 2 ml of molten top agar. The contents of the tube were poured onto minimal glucose agar and incubated at 37C for 48 hr. Concurrent negative (solvent alone) and positive controls (2-aminofluorene and a reference cigarette smoke condensate) were performed with all experiments. All testing was done using triplicate plates at each concentration. A sample was considered to be mutagenic if it induced a concentrationdependent increase in revertant number with at least one concentration being at least two times higher in revertants than the solvent control. Slopes were calculated from linear regression lines constructed from the log of the concentration versus revertant number. The numbers expressed for Ames activity represent the slopes expressed as revertants per milligram of cigarette smoke condensate (Doolittle et ai. 1990). Comparisons of the Ames response between cigarette smoke condensates were made using a t test with Bonferroni's adjustment for multiple comparisons at p = 0.05. Sister Chromatid Exchange (SCE) Assay Cell culture for SCE assay. Chinese hamster ovary (CHO) cells were the WBL strain obtained from Hazleton Laboratories. This cell line has an average cycle time of 12 to 14 hr with a modal chromosome number of 21. Cells were maintained on Ham's F-12 medium supplemented with 10% fetaJ calf serum, 1% L-glutamine, and gentamicin or McCoy's 5a culture medium supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin and streptomycin. All cells were maintained at 37C in a humidified incubator maintained at 95% air and 5% CO2. CHO cells of passage 15 or lower were used in the SCE assay. SCE assay of CSC-exposed cells. Approximately one million cells were seeded in T75 flasks in 10 ml of complete McCoy's 5a medium and cultured for approximately 24 hr prior to treatment. SCE assays without metabolic activation were conducted as follows: Cultures were dosed with the CSC test article (0, 10, 25, 50, 75, and 100 /ig/mJ media) for 2.4 hr before 5bromo-2'-deoxyuridine (BrdU) was added at a final concentration of 10 fjM. Cultures were then reincubated for 22.6 hr. The test article was washed from the cells and fresh complete medium with BrdU and Colcemid (0.1 /ig/ml) was added. Cultures were harvested 2.5 hr later, delayed harvest was required for the higher dose levels due to toxicity-related cell cycle delay. Slides were prepared and stained with the fluorescence-plus Giemsa (FPG) technique (Perry and Wolff, 1974; Goto et ai, 1978). TOX1CITY ASSESSMENT OF A NEW FILTERED CIGARETTE SCE assay of whole smoke-exposed cells. Cells were plated in T75 flasks at a density of 1.2 million cells in 20 ml of Ham's F-12 medium with 25 mM Hepes and 10% fetal calf serum and incubated for 18-24 hr. Just prior to whole smoke exposure, BrdU (10 fiM) was added to each flask and triplicate flasks were exposed to each concentration of whole smoke for 1 hr; triplicate control flasks were exposed to HEPA filtered humidified air for each concentration. After the 1-hr exposure, cells were returned to the incubator and incubated for at least 27.5 hr, with Colcemid for the last 2.5 hr. Higher dose levels required delayed harvest due to cell cycle delay. Cells were then harvested and fixed, and at least three slides were prepared from each flask. Slides were stained according to the FPG technique. SCE assay scoring and data analyses. Seventy-five M2 cells were scored per dose (25 per flask) for the whole smoke-exposed assays. Fifty M2 cells were scored per dose (25 per flask) for the CSC-exposed assays. Slides were scored without knowledge of treatment by an independent laboratory. Healy's test for transformation for variation stabilization (Healy, 1968) was performed and, based on the results of this test, the CSC-exposed assays were analyzed using a square root transformation, and data from the whole smoke-exposed assay were analyzed using a log transformation. Regression lines were fit to the transformed data and slope values were obtained for each cigarette under the separate exposure conditions. Regression analyses (SAS, SAS Institute, Inc., Cary, NC) were used to compare the slopes of the regression lines {p < 0.05). Neutral Red Cytotoxicity Assay Cell culture for the neutral red assay. Chinese hamster ovary cells (WBL strain) obtained from Hazleton Laboratories were maintained in Ham's F-12 medium supplemented with 10% FCS, 0.5 ^g/ml gentamicin, and 5% glutamine. Cells were maintained at 37C in a humidified incubator maintained at 95% air and 5% CO2. Cells between passages 10 and 20 were used for the neutral red assay. Neutral red assay of CSC-exposed cells. The neutral red cytotoxicity assay for determining viable cells was adapted from a procedure described by Borenfreund (Borenfreund and Puemer, 1984, 1985) and modified in our laboratory (Bombick and Doolittle, 1995). The method is described briefly as follows: CHO cells were plated into a 96-well tissue culture plate at a density of 10,000 cells (in 200 /JI of medium) per well. Tissue culture plates were incubated at 37C and 95% air and 5% CO2. After 24 hr the medium was aspirated off and CSC dissolved in DMSO was added to fresh medium to a final 200-//1 volume. The final concentration of DMSO never exceeded 1.5% of the final volume. At the end of the exposure period the medium was aspirated out of each well. Immediately, 200 /jl of a working neutral red solution (1.5 ml of a neutral red stock solution purchased from Sigma Chemical Co. added to 100 ml of F-12 medium without FCS) was added to each well and incubated at 37C for 3 hr in a tissue culture incubator. The neutral red solution was aspirated and 200 fi\ of wash/fix solution (10 ml formalin to 1 liter deionized water) was added to each well for 1 min at room temperature. The wash/fix solution was aspirated off and 100 /il of the solvent solution (10 ml of glacial acetic acid to 50% ethanol) was added to each well. The tissue culture plate was placed on a microplate shaker for 10 min and the absotbance of each well was measured at 540 nm on a microplate reader (Molecular Devices). Neutral red assay of whole smoke-exposed cells. The neutral red assay for determining viable cells after whole smoke exposure has been detailed previously (Bombick etal., 1991, 1995a) and is described briefly as follows: CHO cells were plated into T75 tissue culture flasks at a density of 500,000 cells per flask. Each flask contained 20 ml of medium supplemented with 1 M Hepes buffer to give a final concentration of Hepes in the medium of 25 mM. Cells were maintained for approximately 24 hr in a tissue culture incubator at 37C prior to mainstream whole smoke exposures of I-hr duration. The tissue culture medium was not changed after the cigarette smoke exposures and cells were returned to the tissue culture incubator for approximately 24 hr. The cells were then washed once gently with 20 ml of Hank's balanced salt solution without phenol red (JRH Biosciences, Lenexa, KS). After removing the salt solution wash, 20 ml of a working solution of neutral red was added to the cells in the tissue culture flask and incubated for 3 hr in a tissue culture incubator. The neutral red solution was then aspirated and the cells were fixed quickly with 20 ml of the wash/ fix solution. The wash/fix solution was aspirated and the neutral red solution was extracted from the cells by adding 20 ml of the solvent solution. Aliquots of the neutral red solution from each flask were read in a spectrophotometer (Beckman Instruments DU-70) at 540 nm and the absorbance was recorded. Neutral red assay data analyses. Absorbance averages and standard deviations were obtained for each concentration tested and probit regression analysis was utilized to determine EC50 values. Comparisons between cigarette types were done by comparing concentration-response curves using ANOVA followed by Scheffe's test at a 95% confidence level. In addition, linear regression lines of probit of the response versus the log concentration were constructed (Gad and Weil, 1986; Salsburg, 1986; Finney, 1971). The regression lines were used to calculate EC50 values (i.e., the concentration of cigarette smoke required to elicit a 50% decrease in the neutral red response) and the EC50 values compared. RESULTS The toxicity of the mainstream smoke from the new cigarette containing the new carbon filter and low-nitrogen tobacco blend (EXP-C) was compared to two other experimental control cigarettes [one containing the new carbon filter with a commercial tobacco blend (STD-C) and the other containing the low-nitrogen tobacco blend with a commercial cellulose acetate filter (EXP)] and three commercially available cigarettes (two commercial full-flavor, low-tar cigarettes and a full-flavor, low-tar cigarette with a charcoal filter). The designations and FTC tar, CO, and nicotine values for these six cigarettes are given in Table 1. In vitro toxicity was assessed utilizing two measures of genotoxicity (bacterial mutagenicity and sister chromatid exchange assays) and a measure of cytotoxicity (neutral red assay). A similar approach has been used to examine the cytotoxic and genotoxic potential of another cigarette which primarily heats tobacco (Bombick et al., in press). For detecting the bacterial mutagens in cigarette smoke, the mutagenicity of mainstream CSC was evaluated in Salmonella typhimurium strain TA98 with S9 metabolic activation, the most sensitive strain and condition (Doolittle et al., 1990). The results are shown in Table 2 with data expressed as both revertants per milligram of tar and revertants per cigarette. The CSCs from the two low-nitrogen experimental cigarettes (EXP-C and EXP) had reduced bacterial mutagenicity compared to the STD-C cigarette smoke condensate alone, indicating that tobacco nitrogen content was more important than filter type for modifying the mutagenicity of CSC. Smoke from the six cigarettes was tested in the SCE assay without S9 metabolic activation since in contrast to the Ames test, SCE assays without S9 metabolic activation are reported to be more sensitive to cigarette smoke-related chemicals than SCE assays with S9 activation (Bombick et al., 1995b; Experimental low-nitrogen blend with new carbon filter Experimental low-nitrogen blend with a cellulose acetate Commerical blend with new carbon filter Commercial full-flavor low tar Commerical full-flavor low tar Commerical full-flavor low tar with charcoal filter Doolittle et al., 1990). Figure 1 depicts the induction of SCEs/cell with increasing concentration of CSC from the six cigarettes (expressed as fig tar/ml) without an S9 metabolic activation mixture. There was no difference between any of the six cigarette smoke condensates in induced SCE frequency. Mainstream whole smoke (vapor phase and particulate phase) from the six cigarettes also was examined in the SCE assay and results are reported in Fig. 2. The mainstream whole smoke from the two cigarettes containing the new carbon filter (EXP-C and STD-C) had significantly less potential to induce SCEs when compared to the mainstream whole smoke of the other four cigarettes. Cytotoxicity, as measured by the neutral red assay of the CSC or mainstream whole smoke from the six cigarettes of the study, is reported in Table 3 as calculated EC50 values from probit regression analyses of the concentration-response curves for the cytotoxicity of CSC and mainstream whole smoke. Comparison of the EC50 values of the six Cigarette Smoke Condensate of Mainstream Smoke from the Six Cigarettes Utilized in This Study Cigarette type Revertants/mg tar Revertants/cigarette Note. The Salmonella typhimurium strain TA98 with an S9 metabolic activation system was utilized to examine the potential of the cigarette smoke condensates to induce mutations. Revertant numbers were expressed per milligram of tar and per cigarette. An asterisk denotes statistical significance (p < 0.05) from the smoke condensate of the cigarette containing the novel carbon filter and the experimental low-nitrogen tobacco blend (EXP-C). FIG. 1. SCE response with increasing concentration of cigarette smoke condensates from the six cigarettes studied without metabolic activation. SCE response is reported as mean SCE number ( standard error) per M2 cell (X cells scored) and cigarette smoke condensate concentration is reported as \i% "tar" per milliliter of medium. No statistically significant differences were observed. CSCs indicates no significant statistical differences between any of the CSCs (p > 0.05). However, the mainstream whole smoke from the EXP-C and STD-C cigarettes had significantly lower cytotoxicity (indicated by higher EC50 values) compared to the mainstream smoke from the remaining four cigarettes in the study (p < 0.05). For each cigarette type, the combined mass of 11 chemicals in the vapor phase (ammonia, acrylonitrile, acetone, acrolein, formaldehyde, acetaldehyde, benzene, NOX, HCN, 1,3-butadiene, and isoprene) was correlated to the calculated EC50 values of cytotoxicity and to the slope of SCE concentration-response curve using whole smoke from the six study cigarettes (Fig. 3). There was a statistically significant correlation (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) between biological activity and vapor-phase chemistry. The two cigarettes containing the new carbon filter (EXP-C and STD-C) contained the lowest concentraTOXICTTY ASSESSMENT OF A NEW FILTERED CIGARETTE 2 3 4 5 Cigarette equivalents/cubic meter FIG. 2. SCE response with increasing concentration of whole smoke from the six cigarettes studied. SCE response is reported as mean SCE number (standard error) per M2 cell (X cells scored) and whole smoke concentration is reported as cigarettes per cubic meter. No metabolic activation was utilized for whole smoke SCE assays. An asterisk denotes a statistically significant slope difference of a curve from the slope of all curves without an asterisk (p < 0.05). tions of the 11 biologically active vapor-phase constituents and exhibited the lowest potential to induce cytotoxicity and SCEs. A new cigarette (EXP-C) combining the technology of a new carbon filter with a low-nitrogen tobacco blend has reduced biological activity as measured in several in vitro tests when compared to the commercially available cigarettes tested. The in vitro toxicological assessment of the cigarettes utilized in this study included two measures of genotoxicity and one measure of cytotoxicity. Genotoxic potential of the tobacco smoke was measured by the Ames bacterial mutagenicity assay and the mammalian sister chromatid exchange assay. Both of these assays have been used to measure the genotoxic potential of cigarette smoke (Doolittle et al, 1990). The neutral red assay was utilized to measure the cytotoxic potential of cigarette smoke in this study. The neutral red cytotoxicity assay has been reported to be sensitive in measuring the cytotoxic potential of cigarette smoke (Bombick et al, 1991, 1995a). In vitro assays have traditionally utilized cigarette smoke condensates to examine the cytotoxic and genotoxic potential of cigarette smoke; however, cigarette smoke condensates contain primarily the paniculate phase of cigarette smoke. In this study, we have assessed cigarette smoke condensate for potential genotoxicity and cytotoxicity, but we have also assessed whole smoke utilizing CSET which permits exposure of mammalian cell cultures to whole smoke. CSET has been shown to be a sensitive system for measuring the genotoxicity and cytotoxicity of the vapor phase of cigarette smoke (Bombick et al., 1991, 1995a, in press). Ames bacterial mutagenicity was assessed only utilizing cigarette smoke condensates since the vapor phase of cigarette smoke does not appear to contribute to mutagenicity in this assay (Bombick et al., 1991). Low-nitrogen tobacco blends have been reported to result in the reduction of some biologically active aromatic nitrogen compounds in tobacco smoke (Hoffmann et al, 1980; Schmeltz and Hoffmann, 1977; Benner et al, 1968) and low-nitrogen tobacco blends have exhibited reduced mutagenicity in bacteria compared to higher nitrogen tobacco blends (Munoz et al, 1968; Mizusaki et al, 1977). The low-nitrogen tobacco blend used in this study reduced mutagenic potential in the Salmonella strain TA98 compared to the experimental cigarette in the study containing a standard commercial tobacco blend. However, the low-nitrogen tobacco blend used in this study had no effect on the clastogenicity or cytotoxicity of the condensate when compared to condensate from the cigarette in the study containing a standard commercial tobacco blend. The novel carbon filter has been designed primarily to remove vapor-phase constituents of tobacco smoke with greater efficiency than the charcoal niters of cigarettes currently in the market. Since many of the vapor-phase compounds found in cigarette smoke are genotoxic and cytotoxic in in vitro bioassays (Bombick et al, 1995; GrafStrom et al, 1986; Harris et al, 1985; Saladino et al, 1985), reductions in vapor-phase compounds could have a significant impact on reducing the cytotoxicity and genotoxicity of mainstream smoke. The biological activity of whole smoke in the SCE and neutral red cytotoxicity assays was significantly reduced in cigarettes containing the novel carbon filter (EXP-C and STD-C) compared to the other cigarettes tested. An examina TABLE 3 EC50 Values (Std Dev) of Cytotoxicity for Cigarette Smoke Condensate or Whole Smoke from the Six Cigarettes Utilized in This Study CSC EC50 (tig, CSC/ml) Whole smoke EC50 (cigarettes/cubic meter) Note. The EC50 values for cigarette smoke condensate are expressed as fig CSC per milliliter. The EC50 values for the whole smoke are expressed as the smoke generated by the given number of cigarettes per cubic meter. An asterisk denotes that the EC50 value was significantly different (p < 0.05) from all other cigarettes with the exception of the standard cigarette with the novel carbon filter (STD-C). 1 BOO Mos3 of Irritont Compounds (ug/cigarette) 800 1000 1200 1400 1600 1800 Mn33 of Irritant Compounds (ug/cigarette) tion of the correlation between a measure of vapor-phase chemistry (aggregate mass of 11 major chemicals found in the vapor phase) and the EC50s for cytotoxicity or the slopes of the SCE response curves of the whole smoke from the six cigarettes supports the hypothesis that reductions of vapor-phase components by the new carbon filter are responsible for the reduced cytotoxicity and genotoxicity of the whole smoke (Fig. 3). In conclusion, our results indicate that as measured by the in vitro bioassays utilized, substantial reductions in the biological activity of both the particulate and vapor phases of tobacco smoke can be achieved by combining the novel carbon filter with a low-nitrogen tobacco blend. ACKNOWLEDGMENTS The authors express their thanks to Hema Murli and Carol Spicer at Hazleton Laboratories for the SCE analysis of the cigarette smoke condensates and scoring of prepared SCE slides. The authors also express their gratitude to Jim Com, Mark Higuchi, Shirley Penn, and Lynn Shiels for their technical assistance. The authors acknowledge the contribution of the Analytical Chemistry Division for providing chemical analysis data. Ayres, P. H., Mosberg, A. T., and Coggins, C. R. E. (1990). Modernization of nose-only smoking machines for use in animal inhalation studies. /. Am. Coll. Toxicol. 9, 441-446. Baumgartner, H., and Coggins, C. R. E. (1980). Description of a continuoussmoking inhalation machine for exposing small animals to tobacco smoke. Beitr. Tabakforsch. Int. 10, 169-174. Benner, J. F., Burton, H. R., and Burdick, D. (1968). Composition of cigarette smoke from high and low nitrate burley tobacco. Tob. Sci. 12, 37.


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D. W. Bombick, B. Reed Bombick, P. H. Ayres, K. Putnam, J. Avalos, M. F. Borgerding, D. J. Doolittle. Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter, Toxicological Sciences, 1997, 11-17, DOI: 10.1093/toxsci/39.1.11