ARTICLE IN PRESS

0940-2993/$ - se

doi:10.1016/j.et

�CorrespondiE-mail addr

Experimental and Toxicologic Pathology 62 (2010) 117–126

www.elsevier.de/etp

A summary of toxicological and chemical data relevant to the

evaluation of cast sheet tobacco

Ryan J. Potts�, Betsy R. Bombick, Daniel R. Meckley,Paul H. Ayres, Deborah H. Pence

Research and Development, R. J. Reynolds Tobacco Company, Bowman Gray Technical Center, P. O. Box 1487,

Winston-Salem, NC 27102, USA

Received 19 November 2008; accepted 23 February 2009

Abstract

A tiered testing strategy based on a comparative chemical and biological testing program has been developed toevaluate the potential of tobacco processes, ingredients, or other technological developments to change the biologicalactivity that results from burning tobacco. Cast sheet tobacco is a specific type of reconstituted tobacco sheet that canbe used in the manufacture of cigarettes. The comparative chemical and biological testing program was used tocompare the mainstream smoke and cigarette smoke condensate (CSC) from a Reference cigarette that did not containcast sheet to that collected from Test cigarettes containing cast sheet at a final blend level of either 10% or 15%.Testing included mainstream cigarette smoke chemistry studies, in vitro studies (Ames assay, sister chromatid exchangeassay, and neutral red cytotoxicity assay), and in vivo toxicology studies (13-week rat nose-only inhalation assay and30-week mouse dermal tumor promotion assay). Certain statistically significant differences were observed in thechemical and biological studies when the Reference cigarette was compared to each of the Test cigarettes. However,when viewed collectively, the chemical and biological studies demonstrated that inclusion of cast sheet up to 15% inthe final blend did not increase the inherent biological activity of mainstream cigarette smoke or CSC.r 2009 Elsevier GmbH. All rights reserved.

Keywords: Cast sheet tobacco; Cigarette smoke; Reconstituted tobacco

1. Introduction

Modifications to cigarette design have historicallyinvolved the incorporation of new ingredients, tobaccoprocesses, papers, and filters that have the potential tomodify the quantity and quality of smoke yielded fromcigarettes. The production of reconstituted tobaccosheet (RTS) is an example of a tobacco process widely

e front matter r 2009 Elsevier GmbH. All rights reserved.

p.2009.02.121

ng author. Tel.: +1336 7411930; fax: +1336 7284941.

ess: pottsr@rjrt.com (R.J. Potts).

utilized by cigarette manufacturers as a means to utilizetobacco that cannot be incorporated directly intocigarettes, such as tobacco dust and leaf scraps, as ameans to provide product differentiation (Browne, 1990;Borgerding et al., 1999), and to reduce Federal TradeCommission (FTC) tar and nicotine yields (Hoffmannet al., 1980). Historically, RTS has been included incigarettes at levels up to 25% of the tobacco mass(Browne, 1990). Reconstitution processes result in someloss of natural constituents of the tobacco, yielding adifferent chemical composition compared to the startingtobacco (Norman and Jones, 1998).

ARTICLE IN PRESSR.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126118

RTS can be manufactured either by a papermakingprocess or by a cast sheet (or slurry) process. In theconventional papermaking process, the water-solublefraction of the tobacco scrap is first extracted, leavingbehind a tobacco pulp. The resulting pulp is mechani-cally beaten to fibrillate the cellulose and reduce its fiberlength. The refined cellulose is then formed into a webon the wire screen of a standard papermaking machineand dried by suction and hot air. In a parallel operation,the tobacco extract is concentrated and flavors andhumectants may also be added. The cellulose web isimpregnated with the concentrated extract and thendried.

To manufacture cast sheet, raw tobacco materials areblended and milled to a fine particle size and mixed witha hot aqueous solution of flavors and humectants toform a slurry. Ammoniated compounds are added to theslurry, which function to improve smoke quality andliberate pectin from the tobacco matrix. The releasedpectin acts as a binder to allow the tobacco to form asheet. The slurry is then ‘‘cast’’ onto an imperviousstainless steel conveyor at a controlled rate forming acontinuous sheet of tobacco. The conveyor then travelsthrough a series of heat zones to dry the sheet.

The objective of this document is to summarize andinterpret the chemical and toxicological studies thathave been conducted to evaluate the use of cast sheettobacco in the manufacture of cigarettes. The evaluationincluded mainstream cigarette smoke chemistry studies,in vitro toxicology studies (Ames, sister chromatidexchange, and neutral red cytotoxicity assays), andin vivo toxicology studies (13-week rat nose-onlyinhalation assay and 30-week mouse dermal tumorpromotion assay). The results of the 13-week inhalationassay have previously been reported (Potts et al., 2007)and are not, therefore, duplicated in this manuscript.

2. Materials and methods

2.1. Cigarette description

R.J. Reynolds Tobacco Company sought to substi-tute, in a commercially available product, conventionalRTS created by the papermaking process with cast sheetat up to 15% of the final blend. In order to test thepotential impact of this substitution on the biologicalactivity of cigarettes, a Reference cigarette was com-pared to two Test cigarettes. The Reference cigaretteused a commercial tobacco blend that containedconventional RTS at a level of 30% of the final blend;this cigarette did not contain any cast sheet. The Testcigarettes utilized the same commercial blend as theReference cigarettes except that cast sheet tobacco wassubstituted for a portion of the conventional RTS at two

levels: 10% and 15% of the final blend. All other aspectsof the Reference and Test cigarettes, i.e., paper, filters,ventilation, were the same.

2.2. Mainstream cigarette smoke chemistry study

Selected mainstream cigarette smoke constituentyields were determined by standard methods (Borgerdinget al., 1998; Chepiga et al., 2000). Cigarettes weremachine-smoked under FTC smoking conditions (35mlpuff volume, 2 s duration, 1 puff/min) according to themethod previously described (Pillsbury et al., 1969).Mean analyte values for the two test cigarettes were eachcompared to the control cigarette using a z-test. Thevariability of the methods was estimated from analyticalmonitor data for each of the methods. The standarddeviation measured from the analytical monitor cigar-ette takes into account within day and day-to-daymethod variability and the number of replicate analysesconducted on the sample. p-Values were adjusted usingthe Bonferroni method to control experiment-wise error,with po0.05 required for statistical significance.

2.3. In vitro studies

2.3.1. Preparation of CSC

CSC was prepared by smoking the cigarettes on asmoking machine under FTC conditions as describedabove. Total particulate matter (TPM) was collectedonto Cambridge filter pads. The pads were extractedwith dimethyl sulfoxide (DMSO) to yield a concentra-tion of 10mg ‘‘tar’’/ml DMSO. The CSC samples werestored at �70 1C until assayed.

2.3.2. Mainstream cigarette smoke exposures

Mainstream cigarette smoke exposures were per-formed using cellular smoke exposure technology(CSET) (Bombick et al., 1998a). Mainstream cigarettesmoke was generated by smoking the cigarettes on a30-port rotating carousel (Baumgartner and Coggins,1980), diluting it with humidified HEPA and charcoal-filtered air and distributing it to a two-tier exposurechamber (Cannon et al., 1983). Concentration withinthe chamber was controlled by a feedback-loop ex-posure control system, similar in concept to thatdescribed by Ayres et al. (1990) and verified bygravimetric determination of TPM. Fitted aluminumblocks wrapped with heating tape were used to holdtissue culture flasks on a rocking platform and tomaintain the temperature of the flasks at 37 1C using aprogrammable temperature regulator. Individual cultureflasks were connected to the chamber ports by food-grade silicone tubing and cells within the flask wereexposed to mainstream cigarette smoke by drawingsmoke from the smoke exposure chamber through the

ARTICLE IN PRESSR.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126 119

flask at a flow rate of 275ml/min. Negative controlsutilized humidified HEPA-filtered air drawn through theflask at a flow rate of 275ml/min. The rocking platformallowed the cells to oscillate between exposure tomainstream cigarette smoke and immersion in culturemedium at an approximate rate of 7 cycles/min.

2.3.3. Ames mutagenicity assay

Mutagenicity was assessed in the Salmonella/micro-some assay (Maron and Ames, 1983) with the pre-incubation modification (Yahagi et al., 1975) usingSalmonella typhimurium strains TA98 and TA100 in thepresence of S9 liver homogenate. These strains wereused since they provide the most sensitive, consistentresponse to mainstream CSC (Avalos et al., 2001). S9was purchased from MolTox (Boone, NC, USA) andprepared according to Ames et al. (1975) from maleSprague-Dawley rats that were given a single 500mg/kginjection, i.p., of Aroclor 1254. CSC mutagenicity wasevaluated in the presence of S9 only because, histori-cally, CSC mutagenicity is minimal without metabolicactivation (Bombick et al., 1998b). The S9 concentrationin the S9 mix was 5% (v/v).

CSC sample, S9 mix (0.5ml) and test bacteria (0.1ml)were added to a sterile glass tube. The mixture wasincubated with shaking at 37 1C for 20min prior to theaddition of 2ml of molten top agar containing histidine/biotin. The contents of the tube were mixed and pouredonto minimal glucose agar plates, and incubated at37 1C for approximately 48 h. Concurrent solvent andpositive controls (2-aminoanthracene and a historicalcontrol CSC) were included with all assays. All testingwas done using triplicate plates at each concentration.

Ames data analysis: A sample was considered to bemutagenic if it induced a concentration-dependentincrease in revertant number with at least one concen-tration being at least twice the solvent control. Estimatesof mutagenicity were obtained using the linear point-rejection method of Bernstein et al. (1982).

2.3.4. Sister chromatid exchange (SCE) assay

CSC-exposed cells: SCE assays of CSC-exposed CHOcells were conducted according to established proce-dures (Bombick et al., 1998b) using modifications of theprotocol of Galloway et al. (1985) and Perry and Wolff(1974). In the SCE assay without metabolic activation,CSC was added to the flasks and bromodeoxuridine(BrdU) was added 2 h after beginning of exposure. CSCwas rinsed off the cells after overnight exposure andfresh media with BrdU added. Colcemid was added tothe cultures 2.5 h before harvest. In the assay withmetabolic activation, cells were exposed to the CSC inserum-free media for 3 h in the presence of a rat liver S9reaction mixture (15 ml S9/ml, 1.5mg NADP+/ml, and2.7mg isocitric acid/ml). After exposure, cells werewashed twice and complete medium with BrdU was

added. Cultures were incubated for at least 25.5 h, withcolcemid added for the last 2.5 h. Higher dose levelsrequired delayed harvest due to cell cycle delay underboth activation conditions. Cells were then harvested,fixed, and stained. The positive control agents used inthese assays were mitomycin C for the non-activationseries and cyclophosphamide for the metabolic activa-tion series.

Mainstream cigarette smoke-exposed cells: CHO cellswere plated into T75 flasks at a density of 1.2 millioncells per flask, in 20ml medium supplemented with25mm HEPES buffer and 10% FCS, and incubated for18-24 h at 37 1C in 95% air and 5% CO2. Just prior tosmoke exposure, BrdU (10 mm) was added to each flask.Triplicate flasks were exposed to each concentration ofmainstream cigarette smoke for 1 h. In addition, foreach mainstream smoke concentration, separate tripli-cate flasks were exposed to HEPA-filtered humidified airto serve as controls. Cells were returned to the incubatorand incubated for at least 27.5 h, with colcemidadded for the last 2.5 h. Higher dose levels requireddelayed harvest due to cell cycle delay. Cells were thenharvested and fixed. Slides were stained according to theFPG technique (Perry and Wolff, 1974). The positivecontrol agent used in this assay was mitomycin C.

SCE data analysis: Data were analyzed using regres-sion analysis after square root transformation of thedata (Healy, 1968). Slope values were obtained for eachcigarette (CSC and mainstream cigarette smoke).Regression analyses were used to compare the slopesof the regression lines.

2.3.5. Neutral red cytotoxicity assay

Cytotoxicity in the neutral red assay is evaluated byquantifying the ability of CHO cells to incorporateneutral red dye into the lysosome of the cells; injured orgrowth-inhibited cell cultures exhibit a decreased abilityto incorporate neutral red (Borenfreund and Puerner,1985).

CSC-exposed cells: Neutral red cytotoxicity assays ofCSC-exposed cells were conducted according to themethod of Borenfreund (Borenfreund and Puerner,1984, 1985) as modified in our laboratory (Bombickand Doolittle, 1995; Bombick et al., 1997). CHO cellswere plated in 96-well tissue culture plates at a density of10,000 cells/well. Plates were incubated at 37 1C and95% air/5% CO2 for 24 h. The medium was aspiratedoff and CSC was added to fresh medium to a final 200 mlvolume. Plates were incubated for 24 h. At the end of theexposure period the medium was replaced by 200 ml of a1.5% neutral red solution (Sigma Chemical Co.,St. Louis, MO, USA) combined with medium. Plateswere incubated for 3 h in a tissue culture incubator. Theneutral red solution was removed and 200 ml of aformalin solution was added for 1min, aspirated, andreplaced with an acetic acid/ethanol solution. The tissue

ARTICLE IN PRESSR.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126120

culture plate was placed on a microplate shaker for10min and the absorbance of each well was measured at540 nm on a microplate reader (Molecular Devices,Sunnyvale, CA, USA).

Mainstream cigarette smoke-exposed cells: Neutral redassays of CHO cells exposed to mainstream cigarettesmoke were conducted as previously described(Bombick et al., 1997; Bombick et al., 1998a). CHOcells were plated into T75 flasks at a density of 500,000cells per flask in 20ml medium supplemented withHepes buffer, and incubated at 37 1C and 95% air and5% CO2 for 2472 h. Eight flasks were exposed to eachconcentration of mainstream cigarette smoke for 1 h.In addition, for each mainstream smoke concentration,4 flasks were exposed to HEPA-filtered air to serve ascontrols. After the exposure, cells were returned to theincubator and incubated for approximately 24 h. Thecells were then washed once with balanced salt solution.20ml of a working solution of 1.5% neutral red wasadded and cells were incubated for 3 h in a tissue cultureincubator. The neutral red solution was aspirated andcells were fixed with 20ml of the wash/fix solution. Thewash/fix solution was aspirated and the neutral redsolution was extracted from the cells by adding 20ml ofthe solvent solution. Aliquots of the neutral red solutionfrom each flask were read in a spectrophotometer(Beckman Instruments DU-70, Fullerton, CA, USA)at 540 nm.

Neutral red data analyses: Absorbance averages andstandard deviations were obtained for each concentra-tion tested. Linear regression lines of probit of theresponse versus the log concentration were constructed(Bombick et al., 1998a, b). The regression lines wereused to calculate EC50 values (i.e., concentration ofCSC, mg/ml, to cause 50% reduction in growth of cellpopulation or concentration of mainstream cigarettesmoke, number of cigs/m3 air, to cause 50% reduction ingrowth of cell population) and the EC50 valueswere then compared by one-way analysis of variance(ANOVA).

2.4. Dermal tumor promotion study

The capacity for CSC from the Reference and Testcigarettes to promote dermal tumors was evaluated in a30-week dermal application study as previously de-scribed (Meckley et al., 2004). Animals were housed andcared for according to the ‘‘Guide for the Care and Useof Laboratory Animals’’ (National Research Council,1996). The protocol was reviewed and approved by anInstitutional Animal Care and Use Committee prior tothe conduct of the study.

The study involved groups of 40 female SENCARmice initiated with one application of 7,12-dimethylbenz[a]anthracene (DMBA) or acetone. During weeks 2–7,

a ramped dosing regimen was utilized to acclimate themice to their designated CSC doses three times per week.From study week 8 onward, CSC was administeredat 9, 18 or 36mg ‘‘tar’’/dermal application, three timesper week. Cigarettes were smoked under the FTCconditions, condensates were collected via cold trap,and dosing solutions were prepared in acetone/water.Data recorded in this study included mortality, bodyand organ weights, clinical observations, tumor end-points, gross necropsy observations, and histopathol-ogy. Organ weights were determined for lungs, liver,kidneys, brain, adrenals, spleen, and heart. Organsanalyzed histopathologically for all groups includedtreated dorsal skin, untreated inguinal skin, all skinmasses and skin lesions and all gross lesions. Additionaltissues analyzed histopathologically for solvent controland the high condensate exposure groups included:brain, liver, lungs, kidneys, spleen, adrenals, andovaries.

Data were analyzed using statistical tests availablethrough the PATH/TOX computer system (XybionMedical Systems Corporation, Cedar Knolls, NJ,USA). The total number of tumors was analyzed usinga Chi square test. The number of tumor-bearing animalswas analyzed using Fisher’s Exact Test. Tests werecarried out at the po0.05 two-sided level.

3. Results

3.1. Mainstream cigarette smoke chemistry study

Mainstream cigarette smoke constituents analyzed inthe study included compounds selected from the 1989US Surgeon General’s report (US Department of Healthand Human Services, 1989), the IARC Monograph onSmoking and Health (International Agency forResearch on Cancer, 1986), and by the ConsumerProduct Safety Commission, as toxicants considered topose a risk to health from smoking (Table 1).

Inclusion of 10% and 15% cast sheet resulted instatistically significant increases in ‘‘tar’’, TPM, nicotine,ammonia, catechol, phenol, and p- and m-cresol whencompared to the Reference cigarette on a per cigarettebasis. When smoke chemistry yields were evaluatedbased on equalized ‘‘tar’’ yields (Table 2), inclusion of10% and 15% cast sheet resulted in statisticallysignificant increases in ammonia when compared tothe Reference cigarette. Carbon monoxide, carbondioxide, and acetone (15% cast sheet cigarette only)were significantly reduced in the Test cigarettes incomparison to the Reference cigarette when adjustedfor ‘‘tar’’ yield. The actual tobacco blend componentsand ingredients used to manufacture the cast sheet andconventional RTS differed, which may account for the

ARTICLE IN PRESS

Table 1. Mainstream cigarette smoke chemistry of a Reference cigarette in comparison to Test prototypes containing either 10%

or 15% cast sheet. Yields presented on a per cigarette basisa.

Analyte (yield/cigarette) Reference cigarette

(Mean)

10% Cast sheet

(Mean)

15% Cast sheet

(Mean)

S.D.

Monitorb

Total particulate matter (mg/cig) 12.15 13.19� 13.35� 0.49

Nicotine (mg/cig) 0.79 0.87� 0.87� 0.03

‘‘Tar’’ (mg/cig) 9.85 10.69� 10.90� 0.30

Carbon monoxide (mg/cig) 11.35 11.42 11.67 0.38

Carbon dioxide (mg/cig) 38.2 39.0 39.0 0.74

Ammonia (mg/cig) 14.6 21.4� 23.8� 1.25

Acetaldehyde (mg/cig) 619.6 627.2 614.4 52.7

Acetone (mg/cig) 250.1 258.5 251.7 15.3

Acrolein (mg/cig) 64.5 65.8 65.1 7.7

Formaldehyde (mg/cig) 10.9 9.1 8.9 2.1

Catechol (mg/cig) 42.4 46.4� 46.2� 1.70

Hydroquinone (mg/cig) 44.9 47.5 48.1 2.31

Phenol (mg/cig) 8.7 11.0� 10.4� 0.51

p- and m-Cresol (mg/cig) 7.3 8.7� 8.4� 0.50

Hydrogen cyanide (mg/cig) 159.4 183.9 185.6 18.8

Nitrogen oxides (mg/cig) 208.8 213.0 215.1 9.5

Benzo[a]pyrene (ng/cig) 7.28 7.83 7.81 0.73

N-nitrosoanatabine (ng/cig) 106.0 123.7 121.7 18.4

N-nitrosonornicotine (ng/cig) 103.3 123.7 124.3 24.8

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone

(ng/cig)

92.3 94.3 93.7 8.9

�Statistically significantly different from Reference cigarette at po0.05.aValues have been rounded off to the appropriate number of significant figures.bStandard deviation of the monitor cigarette.

Table 2. Mainstream cigarette smoke chemistry of a Reference cigarette in comparison to Test prototypes containing either 10%

or 15% cast sheet. Yields presented on a per mg ‘‘tar’’ basisa.

Analyte (yield/mg ‘‘tar’’) Reference cigarette

(Mean)

10% Cast sheet

(Mean)

15% Cast sheet

(Mean)

S.D.

Monitorb

Nicotine (mg/mg ‘‘tar’’) 0.080 0.082 0.080 0.001

Carbon monoxide (mg/mg ‘‘tar’’) 1.15 1.07� 1.07� 0.015

Carbon dioxide (mg/mg ‘‘tar’’) 3.88 3.65� 3.57� 0.081

Ammonia (mg/mg ‘‘tar’’) 1.48 2.00� 2.18� 0.12

Acetaldehyde (mg/mg ‘‘tar’’) 62.9 58.8 56.4 5.53

Acetone (mg/mg ‘‘tar’’) 25.4 24.2 23.1� 0.52

Acrolein (mg/mg ‘‘tar’’) 6.54 6.16 5.97 0.37

Formaldehyde (mg/mg ‘‘tar’’) 1.11 0.86 0.82 0.25

Catechol (mg/mg ‘‘tar’’) 4.30 4.35 4.24 0.20

Hydroquinone (mg/mg ‘‘tar’’) 4.56 4.45 4.41 0.26

Phenol (mg/mg ‘‘tar’’) 0.88 1.03 0.96 0.11

p- and m-Cresol (mg/mg ‘‘tar’’) 0.74 0.82 0.77 0.07

Hydrogen cyanide (mg/mg ‘‘tar’’) 16.2 17.2 17.0 0.93

Nitrogen oxides (mg/mg ‘‘tar’’) 21.2 20.0 19.7 0.67

Benzo[a]pyrene (ng/mg ‘‘tar’’) 0.72 0.73 0.73 0.07

N-nitrosoanatabine (ng/mg ‘‘tar’’) 10.5 11.5 11.3 1.76

N-nitrosonornicotine (ng/mg ‘‘tar’’) 10.3 11.5 11.6 2.37

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (ng/

mg ‘‘tar’’)

9.2 8.8 8.7 0.85

�Statistically significantly different from Reference cigarette at po0.05.aValues have been rounded off to the appropriate number of significant figures.bStandard deviation of the monitor cigarette.

R.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126 121

ARTICLE IN PRESSR.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126122

observed differences in smoke chemistry yields. Noother statistically significant differences were observedand all analyte levels were within the ranges typical ofcommercially marketed non-menthol, full-flavor light,cigarettes available in the US between 1995 and 2000(Swauger et al., 2002).

3.2. In vitro studies

3.2.1. Ames study

No statistically significant differences were notedbetween the Reference and Test cigarettes using strainTA98 on either a revertants/mg ‘‘tar’’ or revertants/cigarette basis (Table 3). There was no significantdifference in activity using strain TA100 when theReference cigarette and the Test cigarette containing10% cast sheet were compared on either a revertants/mg‘‘tar’’ or revertants/cigarette basis.

The number of revertants/cigarette with strain TA100was significantly higher for the Test cigarette containing15% cast sheet when compared to the Reference CSC.However, when the number of revertants was expressedon a per mg ‘‘tar’’ basis, no statistical difference wasnoted between the cigarettes. This suggests that theincreased mutagenicity observed with strain TA100 on arevertants/cigarette basis for the 15% cast sheet CSCwas due to the higher levels of ‘‘tar’’ yielded by thiscigarette compared to the Reference CSC.

Fig. 1. Sister chromatid exchange (SCE) assay of cigarette

smoke condensate (CSC) samples from Reference and Test

cigarettes without S9 and with S9 metabolic activation. Data

are expressed as mean SCEs/cell7standard error (S.E.). CSC

samples are expressed as mg ‘‘tar’’/ml media.

3.2.2. Sister chromatid exchange (SCE) study

The CSC and mainstream cigarette smoke from theReference and Test cigarettes induced statisticallysignificant concentration-related increases in SCEscompared to solvent control. There was no significantdifference in SCE frequency between the Reference andTest CSCs, either in the presence or absence of S9metabolic activation (Figs. 1 and 2). In addition, afterexposure to mainstream cigarette smoke, there was nosignificant difference in SCE frequency between smokesderived from the Reference and Test cigarettes.

Table 3. Ames assay of cigarette smoke condensate (CSC) from Ref

TA98 and TA100 in the presence of S9 metabolic activation.

Cigarette Revertants/mg ‘‘tar’’

TA98

Reference cigarette 1,588

Cigarette with 10% cast sheet 1,524

Cigarette with 15% cast sheet 1,999

aStatistically significantly different from Reference cigarette at po0.05.

3.2.3. Neutral red cytotoxicity study

The CSC and mainstream cigarette smoke from theReference and Test cigarettes induced statisticallysignificant concentration-related increases in cytotoxi-city compared to solvent control (Fig. 3). Based on the95% confidence interval of the EC50, mainstreamcigarette smoke generated from the Test cigarettecontaining 10% cast sheet was less cytotoxic incomparison to the Reference cigarette and the Test

erence and Test cigarettes using Salmonella typhimurium strains

Revertants/cigarette

TA100 TA98 TA100

815 15,547 7,979

632 16,413 6,810

1,098 22,089 12,133a

ARTICLE IN PRESS

Fig. 2. Sister chromatid exchange (SCE) assay of mainstream

smoke (MS) from Reference and Test cigarettes. Data are

expressed as mean SCEs/cell7standard error (S.E.). MS

samples are expressed as cigarette equivalents/m3.

Fig. 3. Neutral red cytotoxicity assay of cigarette smoke

condensate (CSC) samples and mainstream smoke (MS) from

Reference and Test cigarettes. Data are expressed as percent of

control7standard deviation. CSC samples are expressed as mg‘‘tar’’/ml media. MS samples are expressed as cigarette

equivalents/m3.

R.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126 123

cigarette containing 15% cast sheet. No other statisticaldifferences were observed for the CSC or mainstreamcigarette smoke samples derived from the Reference andTest cigarettes.

3.3. Dermal tumor promotion study

In the 30-week dermal tumor promotion study,several changes (i.e., erythema, desquamation, peelingskin, abrasions, and sores) of the dosed dorsal skin wereobserved during exposure to CSC, although onlyerythema was attributed to the administration of CSCbecause it was observed within the application site in aconcentration-dependent manner and because it wasabsent in the DMBA/acetone control and acetone/acetone control animals. Administration of the 10% and15% cast sheet Test CSCs produced higher incidences oferythema than the Reference CSC and was present forapproximately 4 of 30 weeks. There were dose depen-dent, treatment-related increases in acanthosis andhyperkeratosis of the dosed skin when compared tothe DMBA/acetone control group. However, there wereno statistically significant differences in acanthosis orhyperkeratosis between comparable doses of 10% or15% cast sheet Test CSCs and the Reference CSC.

Administration of the Reference and cast sheet TestCSCs produced no adverse effects on group mean bodyweights, absolute body weight gain or terminal bodyweights. Administration of the Reference and cast sheetTest CSCs produced statistically significant differencesin the mean absolute organ weight and organ weightratio data of several organs when compared to theDMBA/acetone and acetone/acetone control groups.However, there were no biologically significant differ-ences when compared to each other. There were nobiologically significant non-neoplastic changes of theinternal organs between Test and Reference CSCs.

Several parameters were considered when comparingthe tumorigenicity of the Reference and Test CSCsevaluated in this study (summarized in Table 4), namely:(1) the number of tumor-bearing animals, (2) thenumber of tumors produced, and (3) the time betweenthe beginning of treatment and the development oftumors. The predominant neoplastic changes observedupon microscopic examination of dermal masses orlesions in the dosed skin were papilloma and squamouscell carcinoma. Tumors were produced in all Referenceand Test CSC-treated groups. One microscopicallyconfirmed tumor was produced in the DMBA/Acetonecontrol group. No microscopically confirmed tumorswere produced in the acetone/acetone control group.

Statistical analysis of the number of microscopicallyconfirmed dermal tumor-bearing animals by group,when compared to the appropriate control group(acetone/acetone or DMBA/acetone), showed significant

ARTICLE IN PRESS

Table 4. 30-Week dermal tumor promotion study: summary of microscopically confirmed tumors and grossly observed median

time to onset of masses.

Treatment group initiation/promotion TBMa Total number

of tumors

Median time to onset

of masses (weeks)

Acetone/acetone 0 0 –b

DMBA/acetone 1 1 30

DMBA/reference CSC (9mg ‘‘tar’’/appl) 15c 85c 21

DMBA/reference CSC (18mg ‘‘tar’’/appl) 27c 196c 20

DMBA/reference CSC (36mg ‘‘tar’’/appl) 36c 292c 19

DMBA/10% cast sheet CSC (9mg ‘‘tar’’/appl) 9c 46c,d 24

DMBA/10% cast sheet CSC (18mg ‘‘tar’’/appl) 23c 120c,d 23

DMBA/10% cast sheet CSC (36mg ‘‘tar’’/appl) 38c 409c,e 16

DMBA/15% cast sheet CSC (9mg ‘‘tar’’/appl) 12c 63c 26

DMBA/15% cast sheet CSC (18mg ‘‘tar’’/appl) 29c 190c 20

DMBA/15% cast sheet CSC (36mg ‘‘tar’’/appl) 35c 281c 17

Acetone/reference CSC (36mg ‘‘tar’’/appl) 5 10f 26

Acetone/10% cast sheet CSC (36mg ‘‘tar’’/appl) 13f 21e,f 24

Acetone/15% cast sheet CSC (36mg ‘‘tar’’/appl) 9f 11f 24

appl ¼ application (200 ml).aTumor-bearing mice.bNo tumors produced. Value cannot be calculated.cSignificantly greater than DMBA/acetone control, po0.05.dSignificantly less than corresponding dose of DMBA/Reference CSC, po0.05.eSignificantly greater than corresponding dose of DMBA/Reference CSC, po0.05.fSignificantly greater than acetone/acetone control, po0.05.

R.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126124

treatment- and dose–response relationships for bothReference and Test CSC-treated animals. When com-parable Reference and Test CSC treatment groups wereevaluated, no statistically significant differences werenoted in the number of tumor-bearing animals.

Statistical analysis of the total number of microsco-pically confirmed dermal tumors, when compared tothe appropriate control group (Acetone/Acetone orDMBA/Acetone), showed significant treatment- anddose–response relationships for both Reference andTest CSC-treated animals. When comparable doses ofDMBA/10% cast sheet CSC and DMBA/ReferenceCSC were compared, the DMBA/low- and mid-dose10% cast sheet CSC showed statistically significantlyfewer total numbers of tumors than the DMBA/low-and mid-dose Reference CSC groups, while the DMBA/high-dose 10% cast sheet CSC group had significantlygreater total numbers of tumors than the DMBA/high-dose Reference CSC group. This pattern of response didnot follow a dose–response and did not indicate abiologically significant difference between the 10% castsheet CSC and the Reference CSC. When comparabledoses of DMBA/15% cast sheet CSC and DMBA/Reference CSC were compared, no statistically signifi-cant differences were noted.

For the parameter of the median time to onset oftumors, no difference in biological activity was observedbetween Reference and Test CSCs. Based on thebiological responses observed in the dermal tumorpromotion study, the addition of cast sheet to cigarettes

at a level of inclusion of 10% or 15% in the final blenddid not alter the tumor-promoting characteristics ofthe CSC compared to CSC from cigarettes made withconventional RTS.

4. Discussion

The objective of these studies was to evaluate thesmoke chemistry and relative toxicity of mainstreamcigarette smoke and CSC from Test cigarettes contain-ing cast sheet tobacco and a Reference cigarettecontaining conventional RTS.

An extensive chemical and toxicological dataset wasdeveloped to evaluate the potential impact of cast sheettobacco on the biological activity of mainstreamcigarette smoke and CSC compared to cigarettescontaining conventional RTS. Principal components ofthis evaluation included a determination of selectedmainstream cigarette smoke constituent yields, Amesassay, sister chromatid exchange assay, neutral redcytotoxicity assay, a 30-week dermal tumor promotionevaluation of CSC in SENCAR mice, and a 13-weekinhalation study of mainstream cigarette smoke inSprague-Dawley rats. Results of the 13-week inhalationstudy have been previously reported and demonstratedthat substitution of 10% or 15% cast sheet tobacco forconventional reconstituted tobacco sheet did not alterthe inhalation toxicology of the mainstream smoke

ARTICLE IN PRESSR.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126 125

(Potts et al., 2007). Certain statistically significantchanges in mainstream smoke yields were observedbetween the Reference and Test cigarettes in the presentstudy; however, these differences did not translate intochanges in the biological activity of the mainstreamcigarette smoke or CSC.

In summary, these data demonstrate that the additionof cast sheet tobacco to cigarettes up to a final blendlevel of 15% does not produce a differential response inbiological activity compared to the CSC or mainstreamcigarette smoke from Reference cigarettes that containconventional RTS. Collectively, these data demonstratethat the use of cast sheet tobacco as an alternative formof RTS in the manufacture of cigarettes does not alterthe potential toxicity of CSC or mainstream cigarettesmoke. Therefore, the addition of cast sheet tobacco tocigarette tobacco blends up to 15% is acceptable from atoxicological perspective.

References

Ames BN, McCann J, Yamasaki E. Methods for detecting

carcinogens and mutagens with the Salmonella/mamma-

lian-microsome mutagenicity test. Mutat Res 1975;31:

347–64.

Avalos JT, Lawlor TE, Fowler KW, Bombick BR, Doolittle

DJ. A comparison of the effectiveness of Salmonella strains

for detecting mutagenicity of a cigarette smoke condensate.

Environ Mol Mutagen 2001;37(32):19.

Ayres PH, Mosberg AT, Coggins CRE. Modernization of

nose-only smoking machine for use in animal inhalation

studies. J Am Coll Toxicol 1990;9:441–6.

Baumgartner H, Coggins CRE. Description of a continuous

smoking inhalation machine for exposing small animals to

tobacco smoke. Beitr Tabakforsh Int 1980;19:169–74.

Bernstein L, Kaldor J, McCann J, Pike MC. An empirical

approach to the statistical analysis of mutagenesis data

from the Salmonella test. Mutat Res 1982;97:267–81.

Bombick BR, Murli H, Avalos JT, Bombick DW, Morgan

WT, Putnam KP, et al. Chemical and biological studies of a

new cigarette that primarily heats tobacco. Part 2. In Vitro

toxicology of mainstream smoke condensate. Food Chem

Toxicol 1998b;36:183–90.

Bombick DW, Ayres PH, Doolittle DJ. Cytotoxicity assess-

ment of whole smoke and vapor phase of mainstream and

sidestream cigarette smoke from three Kentucky reference

cigarettes. Toxicol Methods 1997;7:177–90.

Bombick DW, Ayres PH, Putnam K, Bombick BR, Doolittle

DJ. Chemical and biological studies of a new cigarette that

primarily heats tobacco. Part 3. In Vitro toxicity of whole

smoke. Food Chem Toxicol 1998a;36:191–7.

Bombick DW, Doolittle DJ. The role of chemical structure

and cell type in the cytotoxicity of low molecular weight

aldehydes and pyridines. In Vitro Toxicol 1995;8:349–56.

Borenfreund E, Puerner JA. A simple quantitative procedure

using monolayer cultures for cytotoxicity assays (HGD/

NR-90). J Tissue Cult Methods 1984;65:55–63.

Borenfreund E, Puerner JA. Toxicity determined in vitro by

morphological alterations and neutral red absorption.

Toxicol Lett 1985;24:119–24.

Borgerding MF, Bodnar JA, Chung HL, Mangan PP,

Morrison CC, Risner CH, et al. Chemical and biological

studies of a new cigarette that primarily heats tobacco.

Part 1. Chemical composition of mainstream smoke. Food

Chem Toxicol 1998;36:169–82.

Borgerding MF, Perfetti TA, Ralapati S. Determination of

nicotine in tobacco, tobacco processing environments and

tobacco products. In: Gorrod JW, Jacob III P, editors.

Analytical determination of nicotine and related com-

pounds and their metabolites. Amsterdam: Elsevier; 1999.

p. 285–391.

Browne CL. The design of cigarettes, vol. 3. Charlotte, NC:

Hoechst Celanase Corporation; 1990.

Cannon WC, Blanton EF, McDonald KE. The flow past

chamber: an improved nose only exposures system for

rodents. Am Hyg Assoc J 1983;44:923–8.

Chepiga TA, Morton MJ, Murphy PA, Avalos JT, Bombick

BR, Doolittle DJ, et al. 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). Food Chem

Toxicol 2000;38:949–62.

Galloway SM, Bloom AD, Resnick M, Margolin BH,

Nakamur F, Archer P, et al. Development of a standard

protocol for in vitro cytogenetic testing with Chinese

hamster ovary cells: comparison of results for 22

compounds in two laboratories. Environ Mutagen 1985;7:

1–51.

Healy MJR. The disciplining of medical data. Br Med Bull

1968;245:210–4.

Hoffmann D, Tso TC, Gori GB. The less harmful cigarette.

Prev Med 1980;9(2):287–96.

International Agency for Research on Cancer. IARC mono-

graphs on the evaluation of the carcinogenic risk of

chemicals to humans, vol. 38. Lyon, France: Tobacco

smoking; 1986.

Maron DM, Ames BN. Revised methods for the Salmonella

mutagenicity test. Mutat Res 1983;113:247–56.

Meckley DR, Hayes JR, Van Kampen KR, Mosberg AT,

Swauger JE. A responsive, sensitive, and reproducible

dermal tumor promotion assay for the comparative

evaluation of cigarette smoke condensates. Regul Toxicol

Pharmacol 2004;39:135–49.

National Research Council. Guide for the care and use of

laboratory animals. Institute of Laboratory Animal Re-

sources, Commission on Life Sciences. Washington, DC:

National Academy Press; 1996.

Norman A, Jones EG. The history of cigarette design. Recent

advances in tobacco science – responding to changes in our

industry, vol. 24. Atlanta, GA: Tobacco Science Research

Conference; 1998 September 13–16.

Perry P, Wolff S. New Giemsa method for the differential

staining of sister chromatids. Nature 1974;251:156–8.

Pillsbury HC, Bright CC, O’Connor KJ, Irish FW. Tar and

nicotine in cigarette smoke. J AOAC 1969;52:458–62.

Potts RJ, Meckley DR, Shreve WK, Pence DH, Ayres PH,

Doolittle D, et al. Comparative 13-week inhalation study of

ARTICLE IN PRESSR.J. Potts et al. / Experimental and Toxicologic Pathology 62 (2010) 117–126126

cigarette smoke from cigarettes containing cast sheet

tobacco. Inhal Toxicol 2007;19:701–24.

Swauger JE, Steichen TJ, Murphy PA, Kinsler S. An analysis

of the mainstream smoke chemistry of samples of the US

cigarette market acquired between 1995 and 2000. Regul

Toxicol Pharmacol 2002;35:142–56.

US Department of Health and Human Services. Reducing the

health consequences of smoking: 25 years of progress.

A Report of the Surgeon General. US Department of

Health and Human Services, Public Health Service, Centers

for Disease Control, Center for Chronic Disease Prevention

and Health Promotion, Office on Smoking and Health.

DHHS Publication No. (CDC) 89-8411, 1989.

Yahagi T, Degawa M, Seino Y, Matsushima T, Nagao M,

Sugimura T, et al. Mutagenicity of carcinogenic azo dyes

and their derivatives. Cancer Lett 1975;1:91–6.