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Inhalation Toxicology, 2009; 21(8): 688–704
RESEARCH ARTICLE
Eect of ltration by activated charcoal on
the toxicological activity of cigarette mainstream
smoke from experimental cigarettes
Charles L. Gaworski1, Heike Schramke2, Joerg Diekmann3, omas J. Meisgen3, Franz J. Tewes3,
Detlef J. Veltel3, Patrick M. Vanscheeuwijck4, Narayanan Rajendran5, Miguel Muzzio5, and
Hans-Juergen Haussmann6
1Altria Client Services, Richmond, Virginia, USA, 2Philip Morris Products S.A., Neuchâtel, Switzerland, 3Philip Morris
Research Laboratories GmbH, Cologne, Germany, 4Philip Morris Research Laboratories bvba, Leuven, Belgium,
5Illinois Institute of Technology Research Institute, Chicago, Illinois, USA, and 6Toxicology Consultant, Roesrath, Germany
Address for Correspondence: Charles L. Gaworski, Altria Client Services, 601 E. Jackson St, Richmond, VA 23219, USA. e-mail: charles.l.gaworski@altria.com
(Received 15 July 2008; revised 13 August 2008; accepted 13 August 2008)
Introduction
Cigarette smoking is a cause of morbidity and mortality from
diseases including lung cancer, chronic obstructive pulmo-
nary disease (COPD), and cardiovascular disease (CVD) (US
Department of Health and Human Services, 2004). e most
eective way of avoiding these risks is not to smoke, but in
the foreseeable future there will be a signicant part of the
population who will continue to smoke (Morgan et al., 2007;
World Health Organization, 2004). For those people, one
possible approach to reducing the risk of disease is to reduce
exposure to tobacco toxicants (US Institute of Medicine,
2001).
Approaches to wide scale smoke constituent reductions
have been attempted in products that primarily heat rather
than burn tobacco (Eclipse Expert Panel, 2000; Patskan &
Reininghaus, 2003). In addition, incorporation of activated
charcoal (AC) in the lter has been shown to be eective
in the ltration of certain mainstream (MS) vapor phase
and semivolatile constituents (Baggett & Morie, 1975;
Williamson et al., 1965; Xue et al., 2002). Several thousand
individual smoke constituents have been identied (Dube
& Green, 1982), and overlapping toxicological activities and
interactions can be expected between the various chemical
classes of MS constituents. No individual MS constituents
ISSN 0895-8378 print/ISSN 1091-7691 online © 2009 Informa UK Ltd
DOI: 10.1080/08958370802406290
Abstract
Activated charcoal (AC) ltration reportedly decreases the yields of smoke vapor phase constituents including
some identied as human carcinogens and respiratory irritants. Non-clinical studies including chemical smoke
analysis, in vitro cytotoxicity and mutagenicity (bacterial and mammalian cells), and in vivo subchronic rat inha-
lation studies were carried out using machine smoking at ISO conditions with lit-end research cigarettes con-
taining AC lters. The objective was to assess whether AC lter technology would alter the established toxicity
prole of mainstream smoke by increasing or decreasing any known toxicological properties, or elicit new ones.
The reduced yield of vapor phase irritants from AC lter cigarettes correlated with markedly decreased in vitro
cytotoxicity and in vivo morphology of the nose and lower respiratory tract. Increased yields of particulate phase
constituents (e.g. polycyclic aromatic hydrocarbons) in AC ltered smoke were noted in comparison to controls
in some studies. The in vitro bacterial mutagenicity of AC ltered smoke particulate preparations was occasion-
ally increased over control levels. Laryngeal epithelial thickness was increased in some rats inhaling AC ltered
smoke in comparison to controls, an eect perhaps related to higher inspiratory ow. When tested under more
intense Massachusetts Department of Public Health smoking conditions, AC lter associated reductions in vapor
phase constituent yields were smaller than those seen with ISO conditions, but the eect on in vitro cytotoxicity
remained.
Keywords: Activated charcoal ltration; cigarette mainstream smoke; chemical analysis; in vitro toxicity testing;
mutagenicity; cytotoxicity; subchronic inhalation; intense smoking conditions
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Activated charcoal ltration of cigarette smoke 689
which may be responsible for smoking-related diseases have
unambiguously been identied to date. Given the complex
etiologic relationship in smoking-related disease, it will be
dicult to establish whether any change to a tobacco prod-
uct, such as the introduction of an AC lter, will indeed result
in reduced disease risk compared to current conventional
products.
Previous experimental studies on the comparative toxico-
logical properties of charcoal vs. conventional lter cigarettes
demonstrated reduced yields of vapor phase constituents,
reduced in vitro cytotoxicity and genotoxicity, and reduced
toxicity in a few in vivo models (Coggins & Gaworski, 2008).
For many of these studies, the amount, type, and activation
grade of the AC available in these lters is not clear. To date,
there has not been a systematic toxicological investigation
using a dened set of AC ltered cigarettes in a battery of
comparative non-clinical studies.
Here we report a series of non-clinical studies to assess
the potential positive or negative eects of AC in the lters
of lit-end cigarettes on the chemical composition and toxi-
cological activity of smoke. ese studies assessed multiple
levels of AC addition, dierent charcoal sources, and two
types of machine smoking conditions. e list of chemi-
cal analytes and non-clinical toxicological assays used in
these studies was similar to those considered useful in the
previous assessment of tobacco ingredients (Roemer et al.,
2002; Rustemeier et al., 2002; Vanscheeuwijck et al., 2002)
and novel products, such as the electrically heated ciga-
rette smoking system (Schramke et al., 2006; Stabbert et al.,
2003; Terpstra et al., 2003; Tewes et al., 2003). e biological
assays included the Salmonella typhimurium reverse muta-
tion assay, the mouse lymphoma thymidine kinase assay,
the mouse broblast neutral red uptake cytotoxicity assay,
and a 90-day rat inhalation study. e objective of the stud-
ies was to assess whether use of large amounts of AC ltra-
tion in experimental lit-end cigarettes would (1) increase
or decrease any known toxicological properties and/or (2)
elicit the appearance of new toxicological properties of MS.
Materials and methods
General design
e basic cigarette designs and studies conducted for
each set of experimental cigarettes are shown in Table 1.
Experimental cigarettes were prepared with either com-
mercially available AC (>90% activation) prepared from
coconut shells or AC beads (>50% activation) prepared from
synthetic polymer-based hydrocarbon sources. e AC was
added in a plug–space–plug conguration where the mate-
rial was contained between sections of a standard cellulose
acetate cigarette lter.
Since standardized machine smoking conditions neces-
sary to generate smoke for in vitro and in vivo toxicology
studies may not necessarily be representative for the aver-
age human smoker or for the variability in the smoking
population (Hammond et al., 2006), and not all possible
human smoking conditions can be reproduced for toxico-
logical testing, we chose two methods to generate smoke.
Some studies were conducted using the International
Organization for Standardization (ISO)/Federal Trade
Commission smoking conditions (ISO, 1991a; US Federal
Trade Commission, 1967), while other studies were
conducted using smoking conditions suggested by the
Massachusetts Department of Public Health (MDPH)
(Massachusetts General Laws Annotated, 1997). ISO con-
ditions comprise a pu volume of 35 ml taken in 2 s, 1
pu/min, with the ventilation holes of the cigarettes left
unblocked according to ISO standard 3308 (ISO, 1991a),
while the MDPH conditions use more intense smoking with
a pu volume of 45 ml taken in 2 s, 2 pus/min, with 50% of
the ventilation holes blocked (Massachusetts General Laws
Annotated, 1997).
Table 1. Overview of study sets and test cigarettes used in these investigations.
Parameter Set (a) Set (b) Set (c)
Intent Eect of AC dose on chemical smoke
composition
Eect of 180 mg AC on
toxicological activity
Eect of alternate smoking conditions
on AC lter eciency
Test cigarette design Plug–space–plugaPlug–space–plugaPlug–space–pluga
Target AC/cigarette 135, 180, 270 mg 180 mg 180 mg
AC type Synthetic polymer-based beads Coconut shell Coconut shell
ISO TPM yieldb6.0–6.2 mg/cigarette 10.7 mg/cigarette 13.5 mg/cigarette
Control cigarette design Manufactured control Manufactured control 2R4F
Target AC/cigarette 0 mg 0 mg 0 mg
ISO TPM yield 5.9 mg/cigarette 12.6 mg/cigarette 12.2 mg/cigarette
Smoking conditions ISO ISO ISO and MDPHc
Study type
Smoke chemistry Yes Yes Yes
In vitro cytotoxicity No Yes Yes
Bacterial mutagenicity No Yes Yes
Mammalian cell mutagenicity No No Yes
Subchronic inhalation toxicity No Yes No
aCavity between cellulose acetate designed to contain AC.
bISO: International Organization for Standardization smoking conditions; TPM, total particulate matter.
cMDPH Massachusetts Department of Public Health smoking conditions.
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690 C. L. Gaworski et al.
Set (a) used synthetic polymer-based AC beads at target
loadings of 135, 180, or 270 mg per cigarette for analytical
chemical characterization of the resulting MS which was
generated using ISO smoking conditions (Table 1). A cel-
lulose acetate (no charcoal) ltered cigarette was used for
comparison.
Set (b) used activated coconut shell charcoal loadings at
a target of 180 mg per cigarette for in vitro and in vivo toxicity
assays. e MS was generated under ISO smoking conditions
(Table 1). A cellulose acetate (no charcoal) ltered cigarette
was used for comparison.
Set (c) used another type of experimental cigarette with a
target of 180 mg activated coconut shell charcoal per cigarette
for a comparison of two machine smoking conditions (ISO
and MDPH) by analytical chemical characterization of the
MS and in vitro toxicity assays (Table 1). e cellulose acetate
ltered (no charcoal) University of Kentucky reference ciga-
rette 2R4F was used for comparison. e Kentucky reference
cigarette is considered representative of an American-blend
cigarette in terms of construction (Diana & Vaught, 1990),
chemical MS composition, and in vitro (Roemer et al., 2004;
Schramke et al., 2006) as well as in vivo toxicological activity
(Patskan et al., 2008).
Study protocols were derived and adapted to this pur-
pose from internationally accepted guidelines. Studies
were conducted under good laboratory practice conditions
(Organization for Economic Co-operation and Development
Environment Directorate, 1998) (one exception: mouse lym-
phoma assay with MS generated under MDPH conditions).
Care and use of the laboratory rats was in conformity with
national and international regulations and recommenda-
tions (Association for the Assessment and Accreditation of
Laboratory Animal Care International, 1991).
Cigarettes and smoke generation
e research cigarettes used for these investigations con-
tained a typical American tobacco blend comprising bur-
ley, bright, oriental, and reconstituted sheet tobaccos. Test
and control research cigarettes were constructed by Philip
Morris USA, Richmond, VA. Cigarettes were conditioned
following ISO standard 3402 (ISO, 1991b) prior to smok-
ing. ey were smoked on either 20- or 30-port automatic
smoking machines (Borgwaldt GmbH, Hamburg, Germany;
Philip Morris Research Laboratories, Cologne, Germany).
Analytical smoke chemistry
MS constituents were measured with validated analyti-
cal procedures, and quantication and detection limits
were calculated from signal-to-noise ratios (International
Conference on Harmonisation, 2005). e selection criteria
for the MS constituents for analysis as well as the analytical
methods used have been described elsewhere by Stabbert
et al. (2003).
Briey, MS constituents were trapped on glass ber lters,
in wash bottles, or on silica gel cartridges, or directly ana-
lyzed. In general, four replicate smoke samples were gener-
ated per cigarette type under each set of smoking conditions
with 10–20 cigarettes smoked for each sample. Several of the
MS traps required to quantitatively capture the selected con-
stituents added considerable ow resistance to the smoke
path, which meant that the pu prole as standardized for
glass ber lter traps could not be maintained. In such cases,
maintaining the correct pu volume and duration was given
the highest priority.
Mammalian cell cytotoxicity
For the cytotoxicity assay, the neutral red uptake assay
(Borenfreund & Puerner, 1985; INVITTOX, 1990) was per-
formed with mouse embryo BALB/c 3T3 cells, as previously
described (Tewes et al., 2003). Briey, total particulate mat-
ter (TPM) was trapped on glass ber lters and extracted
with dimethyl sulfoxide (DMSO). e rest of the MS, which
passed through the same lter, was bubbled through ice-cold
phosphate buered saline (PBS) to trap the water-soluble
portion of the vapor phase. Cells were exposed to the sepa-
rate fractions for 24 hr prior to 3 hrs incubation with neutral
red. Uptake of neutral red was determined photometrically
at 540 nm. ree batches per cigarette type and smoking con-
dition were generated for each MS fraction. For each batch,
eight MS concentrations with 24 replicates each were tested.
e cytotoxic response to the MS exposure was character-
ized by the EC50 value, i.e. the eective MS concentration
that decreased the number of viable cells by 50% relative to
the solvent control. EC50 values were calculated for each of
the three batches. e reciprocal of the EC50 value (1/EC50) is
reported to better represent increasing cytotoxic eects.
Bacterial mutagenicity
For the bacterial mutagenicity assay, a plate incorporation
version of the Salmonella typhimurium reverse mutation
assay (Maron & Ames, 1983; Organization for Economic
Co-operation and Development, 1997a) was performed in a
design adapted to the comparative testing of cigarette smoke
TPM (fewer doses focusing on the linear dose–response
range), as previously described by Tewes et al. (2003). Briey,
TPM was trapped on glass ber lters and extracted with
DMSO. Two batches were generated per cigarette type and
smoking condition. Determinations were performed with
the tester strains TA98, TA100, TA102, TA1535, and TA1537
in the absence and presence of a metabolic activation sys-
tem consisting of the postmitochondrial fraction of the
livers from rats treated with Aroclor 1254 (MA BioReliance
Corporation, Rockville, MD). For each of two TPM batches,
three doses of TPM dissolved in DMSO were prepared and
assayed. Each dose was plated in triplicate. e mutagenic
response reported here was calculated as the slope (rever-
tants/mg TPM) of the linear portion of the dose–response
curve tted with Poisson-weights to the data for each of the
two batches.
Mammalian cell mutagenicity
For the mammalian cell genotoxicity assay, the micro-
titer plate version of the mouse lymphoma thymidine
kinase assay (Organization for Economic Co-operation
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Activated charcoal ltration of cigarette smoke 691
and Development, 1997b) was performed using L5178Y/
tk+/--3.7.2C mouse lymphoma cells (Cytotest Cell Research,
Rossdorf, Germany) in the absence and presence of a meta-
bolic activation system (see above), and adapted to the test-
ing of MS as previously described by Schramke et al. (2006).
Two batches of TPM were produced under ISO smoking
conditions and dissolved in DMSO. For MDPH conditions,
only one batch was generated. e dose–response curve
for the mutant frequency was calculated for each of the
TPM batches individually by non-linear regression analysis
with the power function y = a + bxc. From the dose–response
curves, the equal mutagenic eect concentration for three
times the background (spontaneous) mutant frequency,
or C3B, was calculated. e condence limits for the C3B
values were derived from the 95% condence limits of the
curves.
Subchronic inhalation toxicity
Inhalation toxicity was investigated in a 90-day rat inhala-
tion study using a test protocol (Organization for Economic
Co-operation and Development, 1981) that has been adapted
to investigate and compare systemic and in particular local
respiratory tract eects of MS inhalation. e 90-day rat
inhalation study has previously proved to be useful in inves-
tigating potential modications in the toxicological prole
of MS (Carmines et al., 2005; Lemus et al., 2007; Terpstra
et al., 2003; Vanscheeuwijck et al., 2002). Briey, healthy male
and female Sprague–Dawley rats (Crl:CDBR, Charles River
Deutschland) were nose-only exposed to diluted MS for
6 hours per day, 7 days per week. TPM concentrations were
set to 150 mg/m3. is level has previously resulted in only
moderate nasal histopathology using a 6 h/d, 7 d/w expo-
sure, and therefore allows sensitivity in detecting increased,
as well as decreased, toxicity. To outline the dynamic range
of ndings and for quality control, a sham-exposed group of
rats (exposed to HEPA (high eciency particulate air)-ltered
conditioned air) was included. e age of the rats at the start
of the inhalation period was approximately 6 weeks. Because
the machine used to generate smoke for the inhalation study
was originally designed based on ISO conditions, cigarettes
could only be smoked according to one standard protocol
(ISO, 1991a). To monitor the stability and reproducibility of
the MS exposure, chemical and physical analyses were per-
formed as described previously by Haussmann et al. (1998).
e rats were housed and exposed in an animal laboratory
with restricted access under specied hygienic conditions.
e room temperature was maintained at 20.2°C (SD 0.6)
and the relative humidity at 54.1% (SD 4.0). e light/dark
cycle was 12 h/12 h. Respiratory parameters, representative
nicotine metabolites in urine, blood carboxyhemoglobin
proportions, food consumption, body weight, ophthalmos-
copy, and clinical observations were determined as previ-
ously described (Coggins et al., 1981; Haussmann et al., 1998;
Klimisch et al., 1974; Rustemeier et al., 1993; Vanscheeuwijck
et al., 2002).
Hematology and clinical chemistry parameters (Organi-
zation for Economic Co-operation and Development, 1981)
were determined for all rats at necropsy, without prior fasting
(Vanscheeuwijck et al., 2002). Weights of lungs with larynx
and trachea as well as liver, heart, adrenal glands, testes,
brain, spleen, and kidneys were recorded at necropsy. Lungs
with larynx and trachea were removed together and xed
with EAFS (ethanol–acetic acid–formaldehyde–saline) solu-
tion by instillation at 20 cm water pressure. Tissue lesions
noted at necropsy and major organs were collected and
processed for histopathology using standard techniques.
Special histological sections were prepared for the nose
according to the method of Young (1981), and for the larynx
according to Lewis (1981). e trachea was cut transversally
(near the larynx) as well as frontally (at the bifurcation).
One frontal section passing through the main bronchus
for the left lung and one frontal section passing through a
maximum number of lobes for the right lung were prepared.
Individual sections were 5–6 µm thick and stained routinely
with hematoxylin and eosin. Sections from nose level 1,
tracheal ring and bifurcation, and lungs were additionally
stained with Alcian blue/periodic acid–Schi’s reagent to
facilitate recognition of mucus-producing goblet cells. e
laryngeal epithelial thickness at the oor of the larynx was
also determined morphometrically using a projection of the
microscopic image onto a screen. A veterinary pathologist
with experience in cigarette smoke-related changes in the
respiratory tract of rodents read the slides in a blind man-
ner. Histopathological ndings were scored according to a
dened severity scale from 0 to 5. Mean severity scores were
calculated based on all rats in a group.
Statistical evaluation
For the analytical chemical studies, data were analyzed using
one-way analysis of variance for overall comparison. In case
of a signicant result, pairwise comparisons between each
particular group and the respective control group followed
(Dunnett, 1955). For samples with at least one value below
the limit of quantication, the Kruskal–Wallis test was used.
For cytotoxicity, the mean of the EC50 obtained from three
independent batches was evaluated by pairwise compari-
sons of test cigarettes with and without added AC using the
t-test. For bacterial mutagenicity, pairwise comparisons of
test cigarettes with and without added AC were performed
for each TPM batch separately using the analysis of variance
with continuous-by-class design (analysis of covariance)
(Zar, 1984). e mammalian cell mutagenicity between the
test cigarettes with AC in the lter and the reference ciga-
rette 2R4F was considered to be signicantly dierent if the
95% condence limits of the C3B values did not overlap. For
each TPM batch, a pairwise comparison was carried out. In
an overall evaluation, the mutagenicity between the test and
the reference cigarette was considered to be signicantly dif-
ferent if both comparisons of the two pairs of batches were
signicantly dierent.
For continuous data in the inhalation study, the analy-
sis of variance was applied for overall comparison fol-
lowed by either the Dunnett test for pairwise comparisons
to the sham-exposed group or the Tukey test for pairwise
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692 C. L. Gaworski et al.
comparisons between the MS exposure groups (Dunnett,
1955; Zar, 1984). For ordinal data, the Cochran–Mantel–
Haenszel test (Mantel & Haenszel, 1959) was applied for
overall comparisons and pairwise comparisons.
All tests were conducted at the nominal level of signi-
cance of = 0.05 (two-tailed). Results were considered sta-
tistically signicant at p ≤ 0.05. No adjustment for multiple
testing was made.
Results
Set (a): changes in analytical smoke composition
by adding various amounts of AC in the lter
e introduction of synthetic polymer-based AC beads in
the lter did not substantially alter the basic smoke charac-
teristics as measured by the number of pus and the yields
of TPM, tar (TPM minus nicotine and water), and carbon
monoxide per cigarette (Table 2). Relative to TPM and tar,
nicotine showed a slight decreasing trend with increasing
AC loading.
Smoke constituents mainly found in the vapor phase of
MS were principally aected by AC ltration (Table 2). e
most distinct decrease was between the control and the
lowest level of AC (135 mg), with only modest additional
changes resulting from greater AC addition levels. Notable
were reductions in the yields of 1,3-butadiene (up to –90%
on a per cigarette basis), benzene (up to –87%), isoprene
(up to –88%), acetaldehyde (up to –72%), acrylonitrile
(up to –88%), styrene (up to –89%), acrolein (up to –78%),
hydrogen cyanide (up to –52%), and toluene (up to –87%).
e more polar constituents, such as carbon monoxide or
the nitrogen oxides (mainly nitric oxide), were not reduced.
Formaldehyde, distributed between the vapor and particu-
late phases, was reduced only up to –28%. Cadmium was
reduced up to –52%.
Particulate phase constituents, such as acetamide, aro-
matic amines, tobacco-specic N-nitrosamines, and polycy-
clic aromatic hydrocarbons, as well as arsenic and lead, were
generally unaected by AC addition (Table 2). Some smoke
constituents on the list of analytes could not be detected or
quantied in either of the cigarette types, such as some vola-
tile N-nitrosamines or particulate phase polycyclic aromatic
hydrocarbons, as well as chromium and nickel.
When compared on a TPM relative basis, the smoke
constituent changes in activated charcoal ltered smoke
described above were qualitatively similar, since the addi-
tion of AC did not signicantly modify the TPM yield. Since
the nicotine yield was slightly decreased by AC ltration,
comparison of smoke constituents on a nicotine relative
basis indicated increased yields of up to approximately 20%
for some particulate matter compounds, while decreases
remained for the vapor phase constituents.
Set (b): changes in toxicological activity by adding
180 mg of AC in the lter
e data in this section are presented on a TPM relative basis
in order to allow a comparison of the quality of the MS of the
two types of research cigarettes. Smoke constituent analy-
sis of the control cigarette without AC had a TPM yield of
approximately 13 mg per cigarette, while the TPM yield for
the AC cigarette was about 15% less. us, to estimate the
relative potency of the two types of MS on a per cigarette
level, the data presented on a TPM relative basis need to
be corrected for the 15% lower TPM yield in the test com-
pared to the control cigarette. Analytical smoke chemistry
data indicated that the coconut shell AC added to the test
cigarette used in set (b) was at least as ecient at reducing
vapor phase constituents as the test cigarette type with the
same level of synthetic polymer-based AC used in set (a)
(e.g. about −92% for 1,3-butadiene and acrolein on a per mg
TPM basis). However, some particulate phase constituents
such as 2-naphthylamine were increased by up to +48% on
a per mg TPM basis (+26% on a per cigarette basis), while
some measurable polycyclic aromatic hydrocarbons (PAHs)
increased from 10 to 25% on a per mg TPM basis compared
to the control.
In vitro cytotoxicity
e cytotoxic activity of the TPM fraction did not change
as a result of the AC ltration (1/EC50 value of three inde-
pendent batches per test cigarette (mean ± SE): 0 mg AC,
8.65 ± 0.42 ml/mg TPM; 180 mg AC, 8.53 ± 0.58 ml/mg TPM).
Compared on a per cigarette basis, the cytotoxic activity of
the TPM fraction was slightly lower in the smoke of the AC
ltered cigarette than in that of the control cigarette; how-
ever, this dierence was not statistically signicant.
For the vapor phase fraction of the AC ltered MS, no EC50
value could be determined due to the lack of measurable
cytotoxicity, even though the concentration range tested was
extended by three-fold compared to the control cigarette
(Figure 1). e measured absorbance values at 50% toxicity
corresponded to an average 1/EC50 value of 5.31 ± 0.50 ml/
mg TPM or 69.3 ± 7.2 ml/cigarette in the smoke of the control
cigarette. e signicant reduction of the cytotoxic activity of
the vapor phase of the AC ltered cigarette smoke correlated
Figure 1. Eect of AC ltration on in vitro cytotoxicity of the MS vapor
phase (three independent batches per cigarette type without or with AC).
0
0 100 200 300 400
Dosing concentration (mg TPM/I)
500
0 mg AC-Batch 1
EC50 calculation line
0 mg AC-Batch 2
0 mg AC-Batch 3
180 mg AC-Batch 1
180 mg AC-Batch 2
180 mg AC-Batch 3
600 700 800
25
50
75
100
125
Absorbance Relative to Control (%)
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Activated charcoal ltration of cigarette smoke 693
Table 2. Chemical analysis of MS from cigarettes containing lters with dierent levels of synthetic beads: set (a).
ParameteraUnit/cigarette 0 mg AC (mean ± SD) 135 mg AC (mean ± SD) 180 mg AC (mean ± SD) 270 mg AC (mean ± SD)
ISO parameters
Total particulate matter mg 5.93 ± 0.17 6.06 ± 0.077 6.00 ± 0.19 6.18 ± 0.12
Tar mg 5.05 ± 0.17 5.25 ± 0.081 5.17 ± 0.20 5.35 ± 0.13*
Nicotine mg 0.535 ± 0.020 0.529 ± 0.020 0.515 ± 0.028 0.507 ± 0.013
Water mg 0.341 ± 0.069 0.288 ± 0.050 0.317 ± 0.064 0.323 ± 0.078
Carbon monoxide mg 5.51 ± 0.44 6.06 ± 0.33 5.45 ± 0.56 5.86 ± 0.086
Pu count — 8.0 ± 0.1 7.9 ± 0.14 7.9 ± 0.13 7.8 ± 0.24
Aliphatic hydrocarbons
1,3-Butadiene g20.4 ± 1.4 2.17 ± 0.19* 3.12 ± 0.51* 2.05 ± 0.39*
Isoprene g206 ± 9.2 35.4 ± 12* 35.2 ± 3.7* 23.7 ± 6.0*
Aldehydes
Formaldehyde g5.18 ± 0.52 4.20 ± 0.49* 4.02 ± 0.42* 3.75 ± 0.60*
Acetaldehyde g279 ± 19 106 ± 22* 110 ± 15* 78.4 ± 12*
Acrolein g27.8 ± 2.6 7.52 ± 1.7* 8.68 ± 1.7* 6.08 ± 1.2*
Propionaldehyde g19.4 ± 1.0 5.06 ± 1.6* 5.61 ± 0.69* 3.86 ± 0.55*
Aliphatic nitrogens
Acrylonitrile g6.10 ± 0.45 0.99 ± 0.29* 0.98 ± 0.16* 0.72 ± 0.14*
Hydrogen cyanide g45.9 ± 4.5 25.0 ± 2.9* 21.8 ± 3.3* 22.0 ± 2.9*
2-Nitropropane g10.6 ± 0.72 8.76 ± 1.2* 8.13 ± 0.70* 7.79 ± 0.70*
Acetamide g2.67 ± 0.075 2.37 ± 0.13* 2.54 ± 0.22 2.73 ± 0.16
Aromatic amines
o-Toluidine ng 35.1 ± 2.1 35.9 ± 1.7 40.5 ± 2.4 40.1 ± 4.6
2-Naphthylamine ng 5.05 ± 0.1 5.17 ± 0.29 5.51 ± 0.24 5.41 ± 0.59
4-Aminobiphenyl ng 0.944 ± 0.15 0.953 ± 0.078 0.984 ± 0.075 0.954 ± 0.01
o-Anisidine ng 1.82 ± 0.054 1.87 ± 0.10 2.07 ± 0.055 2.05 ± 0.29
Halogen compounds
Vinyl chloride ng 16.3 ± 3.7 <16b<16b<16b
Inorganic compounds
Nitrogen oxides mg 0.150 ± 0.008 0.146 ± 0.003 0.135 ± 0.010* 0.137 ± 0.007
Monocyclic aromatic compounds
Benzene g23.7 ± 1.4 4.75 ± 1.2* 4.24 ± 0.47* 3.13 ± 0.50*
Toluene g42.9 ± 4.0 9.91 ± 2.5* 7.73 ± 1.1* 5.71 ± 0.99*
Styrene g2.76 ± 0.47 0.658 ± 0.13* 0.383 ± 0.064* 0.313 ± 0.081*
N-nitrosaminesc
N-nitrosodimethylamine ng 1.86 ± 0.38 <1.4b<1.4b<1.4b
N-nitrosodipyrrolidine ng 3.49 ± 0.29 3.52 ± 0.54 3.00 ± 0.24 2.72 ± 0.26*
N-nitrosonornicotine ng 111 ± 5.4 114 ± 7.8 110 ± 4.8 117 ± 4.6
NNK ng 83.9 ± 3.7 85.6 ± 3.5 84.0 ± 3.2 87.7 ± 3.0
Phenols
Phenol g11.2 ± 0.8 9.48 ± 0.31* 9.49 ± 0.23* 9.66 ± 0.72*
Catechol g33.9 ± 1.2 33.9 ± 0.64 32.7 ± 0.36 34.4 ± 1.4
Polycyclic aromatic hydrocarbonsc
Benzo[a]anthracene ng 7.09 ± 0.83 7.44 ± 0.26 6.79 ± 0.85 6.83 ± 0.59
Benzo[b]uoranthene ng 3.40 ± 0.19 3.63 ± 0.066 3.40 ± 0.23 3.57 ± 0.22
Benzo[k]uoranthene ng 1.47 ± 0.17 1.53 ± 0.063 1.49 ± 0.13 1.51 ± 0.16
Benzo[j]uoranthene ng 2.13 ± 0.16 2.13 ± 0.11 2.04 ± 0.15 2.14 ± 0.12
Benzo[a]pyrene ng 4.18±0.23 4.31 ± 0.19 4.09 ± 0.22 4.34 ± 0.29
Indeno(1,2,3-cd)pyrene ng 1.97±0.059 1.99 ± 0.072 1.98 ± 0.12 2.15 ± 0.16
Elementsc
Cadmium ng 28.5±1.7 14.8 ± 0.38* 15.3 ± 1.4* 13.6 ± 0.53*
Arsenic ng 2.51±0.13 2.49 ± 0.079 2.46 ± 0.059 2.49 ± 0.025
Lead ng 6.99±1.40 7.11 ± 0.50 7.38 ± 2.0 7.32 ± 0.84
Note: Asterisk indicates signicantly dierent from the 0-mg AC cigarette, p < 0.05.
an = 4 replicates with 4–20 cigarettes smoked per replicate.
bAt least 1 value below quantitation limit, median shown, SD not calculated.
c e nitrosamines N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, and N-nitrosopiperidine,
the PAHs dibenz[a,h]anthracene, 5-methylchrysene, dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,i]pyrene, dibenzo[a,h]pyrene, and the elements
chromium and nickel were all below the quantitation limit for the assay.
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694 C. L. Gaworski et al.
with a –92% change of the acrolein concentration (measured
by trapping the vapor phase of the two research cigarettes in
buer solution).
Bacterial mutagenicity
As in previous cigarette smoke studies under similar con-
ditions (e.g. Roemer et al., 2002), the TPM was mutagenic
in tester strains TA98, TA100, and TA1537 in the absence
and presence of the metabolic activation system (Table 3).
e other tester strains used responded either negatively
or with only borderline response. With the exception of
a statistically signicant increase (average +24%) in the
mutagenicity of the MS of the AC ltered cigarette com-
pared to the respective control cigarette in tester strain
TA98 in the presence of the metabolic activation system,
no statistically signicant dierences were apparent as
a result of AC addition to the lter of these experimental
cigarettes. A numerical but statistically non-signicant
increase by AC ltration was also observed for tester strain
TA100 in the presence of the metabolic activation system.
On a per cigarette basis, no AC-induced dierence was
observed (data not shown).
Subchronic inhalation toxicity study
On a TPM basis, AC ltration resulted in a reduced nico-
tine concentration (–16%) in the test atmosphere (Table 4),
which was not observed during the MS sample generation
for the in vitro studies with the same research cigarettes.
Aldehyde concentrations were lower (formaldehyde, −73%;
acetaldehyd e, −70%; acrolein, –89%) in the test atmosphere
of the AC ltered cigarettes compared to that of the cellu-
lose acetate ltered control cigarettes. ere was no eect
on the particle size distribution by AC ltration in terms
of mass median aerodynamic diameter and its geometric
standard deviation (i.e. both aerosols were equally respir-
able for the rats).
Table 4. Summary of exposure chamber atmosphere analyses for MS from cigarettes containing AC lters compared to cellulose acetate ltered
controls: set (b).
Analytical parameter Unit nSham control 0 mg AC (mean ± SD) 180 mg AC (mean ± SD)
TPM mg/m393 <0.9 149 ± 5 151 ± 4
Nicotine mg/m315 <0.03 9.9 ± 0.5 8.3 ± 0.6
Carbon monoxide ml/m393 <1.5 129 ± 5 144 ± 6
Formaldehyde ml/m311 — 0.55 ± 0.07 0.15 ± 0.01
Acetaldehyde ml/m311 — 5.6 ± 0.5 1.7 ± 0.1
Acrolein ml/m311 — 0.55 ± 0.06 0.06 ± 0.01
MMAD m2 — 0.40 0.39
GSD m 1.65 1.65
Note: MMAD, mass median aerodynamic diameter; GSD: geometric standard deviation.
Table 3. Bacterial mutagenicity of MS from cigarettes containing AC lters compared to cellulose acetate ltered controls: set (b).
S9 Strain Batch
0 mg AC 180 mg AC
Summary eectb
Revertants/mg TPM
(mean ± SE) Linearity/signicancea
Revertants/mg TPM
(mean ± SE) Linearity/signicance
Without TA98 1 15 ± 3 L/+ 14 ± 3 L/+ =
2 16 ± 4 L/+ 10 ± 6 L/= =
Overall =
TA100 1 116 ± 19 L/+ 114 ± 15 L/+ =
2 133 ± 23 L/+ 132 ± 16 L/+ =
Overall =
TA1537 1 7 ± 2 L/+ 6 ± 1 L/+ =
2 6 ± 2 L/+ 10 ± 1 L/+ =
Overall =
With TA98 1 3296 ± 139 L/+ 3921 ± 62 L/+ *
2 3326 ± 162 L/+ 4290 ± 73 L/+ *
Overall*
TA100 1 1500 ± 115 L/+ 1748 ± 137 L/+ =
2 1593 ± 112 L/+ 1790 ± 98 L/+ =
Overall =
TA1537 1 222 ± 59 N/+ 324 ± 59 L/+ =
2 296 ± 48 L/+ 285 ± 36 L/+ =
Overall =
aLinearity (L) or non-linearity (N) of the dose–response curve/+ indicates dierence in slope of the dose–response curve from zero, p ≤ 0.05.
bPairwise comparisons of test cigarettes with and without activated charcoal by covariance analysis. Asterisk indicates signicant dierence, p ≤ 0.05. =
indicates dierence was not signicant, p > 0.05. Overall eect considers the repeatability of the two batches tested.
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Activated charcoal ltration of cigarette smoke 695
For dosimetry purposes, tidal volume and respira-
tory frequency were measured, with resulting respiratory
minute volume calculated. ese parameters can also be
useful measures of respiratory irritation and, depending
on the irritative potency and/or concentration of the MS
tested, a change (reduction) in the respiratory minute
volume may be expected upon smoke exposure (Terpstra
et al., 2003). Despite expectations, with AC ltration the
changes in respiratory minute volume were similar for
the male rats exposed to the MS of the AC ltered ciga-
rettes (−28% compared to sham-exposed males) and those
exposed to the MS of the cigarettes without AC ltration
(−20%). However, for female rats, there was a 22% reduc-
tion (statistically signicant) for the control group, with no
signicant change (+6%) in the test group, in comparison
to the sham-exposure group. In the female rats, this was
attributable to changes in the respiratory frequency (–19%
for the control and +4% for the test group) and not in tidal
volume. A peak inspiratory ow rate was determined
based on the above measurements. Control MS exposure
signicantly reduced this ow rate by 36% in both genders
(Table 5). While there was no dierence between MS types
for male rats, the peak inspiratory ow rate in female rats
exposed to the MS of the AC ltered cigarettes was signi-
cantly greater than in control MS-exposed female rats, and
similar to that of the sham-exposed female rats. Together,
the observed dierences between the control and AC
ltered MS-exposed groups preclude a clear conclusion
on any potential eect of the AC ltration on the actual
inhaled MS dose.
Biomarkers of exposure were determined for quality
control and to reveal potential dierences in exposure due
to dierences in MS composition between the two test ciga-
rettes. Carboxyhemoglobin proportions in the blood of the
MS-exposed rats varied between 16.8 and 20.2% (largest SE
0.6%, n = 6 for males and females each) for test atmosphere
carbon monoxide concentrations between 124 and 153 ppm.
ere was no relevant dierence between rats exposed to the
two types of MS. Nicotine and nicotine metabolites excreted
in the urine showed a similar relative distribution pattern for
both MS-exposed groups (Figure 2). e total amount excreted
was 50–60% and 60–70% of the theoretical inhaled nicotine
dose in males and females, respectively (using the nicotine
concentrations in the test atmospheres, the daily exposure
duration, and the respiratory minute volume, calculated
according to Guyton, 1947). ese results corresponded well
with the expected 60% recovery calculated in previous studies
(Schepers et al., 1993). ere was no observable inuence by
AC ltration on the uptake or metabolism of nicotine.
Food consumption per unit body weight was similar in all
groups for male and female rats. Body weight increased in
all groups throughout the inhalation period (Figure 3). e
body weight gain in female rats was not dierent whether
exposed to MS or fresh air. MS-exposed male rats had a
reduced body weight development, resulting in signicantly
lower body weights at the end of the inhalation period in
control MS-exposed (−29%) and AC ltered MS-exposed
(−19%) groups compared to the sham-exposed group. e
weight gain in the AC ltered male MS group was signi-
cantly greater than that in the control male MS group (+14%),
but remained lower than sham controls.
One female rat of the control MS-exposed group died
during the study due to technical error. After daily exposure,
decreased turning reex, decreased gripping ability, increase
in irritability (reaction to sound and touch), Harderian gland
AC level (mg)
0180 0180
Relative distribution of excreted
nicotine metabolites (%)
0
20
40
60
80
100
nicotine-N'-oxide
cotinine
3'-hydroxycotinine
3-pyridylacetic acid
4-(3-pyridyl)-4-oxobutyric acid
4-(3-pyridyl)-4-hydroxybutyric acid
nornicotine
5'-hydroxycotinine
nornicotine
Male Female
Figure 2. Relative distribution of nicotine metabolites in male and
female rats. e values represent the percentage of each metabolite rela-
tive to the total recovered in the urine.
Study Day
0 10 20 30 40 50 60 70 80 90 100
Body Weight (gm)
100
200
300
400
500 Male - Sham control
Male - 0 mg AC
Male - 180 mg AC
Female - Sham control
Female - 0 mg AC
Female - 180 mg AC
Figure 3. Body weight development in the subchronic inhalation study.
For clarity of presentation, error bars indicating data variability are
omitted.
Table 5. Peak inspiratory ow in rats exposed to MS from cigarettes
containing AC lters compared to cellulose acetate lter controls.
Gender Unit
Peak ow (mean ± SD)
Sham control 0 mg AC 180 mg AC
Male ml/sec 7.82 ± 0.54 4.99 ± 0.38 4.53 ± 0.58
Female ml/sec 7.65 ± 0.46 4.86 ± 0.54 7.08 ± 0.38*
Note: Bold print indicates statistically dierent from sham-exposed
control (MS inhalation eect). Asterisk indicates signicant dierences
from group exposed to MS from cellulose acetate lter control (0 mg AC),
p ≤ 0.05.
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696 C. L. Gaworski et al.
secretion, yellow-brown discolored, wet fur, changes in
spontaneous activity, and minor wounds on the paws were
observed. e incidences of these ndings were higher in the
MS-exposed groups than in the sham-exposed group, with
no overall dierence in the pattern of these ndings related
to AC ltration. No MS exposure-related ophthalmological
ndings were observed at the end of the inhalation period.
Hematology measurements showed MS-dependent dif-
ferences with regard to the dierential leukocyte count for
both genders (Table 6). Exposure to the MS of the control
cigarette without AC ltration signicantly reduced the lym-
phocyte count (−46% for males, −25% for females) compared
to sham controls. No signicant eect was apparent in the
groups exposed to AC ltered MS (−23% for males, −1% for
females) compared to controls (i.e. a less pronounced eect
following exposure to MS of AC ltered cigarettes). In addi-
tion, there was a trend toward increased blood neutrophil
counts in groups exposed to smoke from either cigarette
type. No eect was observed for red blood cell indices and
platelet counts, with the exception of a trend toward higher
hemoglobin values in MS-exposed rats.
Serum enzyme activities did not reveal any particular
sign of organ toxicity with only a few statistically signicant
increases (e.g. alkaline phosphatase in female rats exposed
to the MS of control cigarettes), or decreases (e.g. serum
protein in MS-exposed male and female rats) (Table 7). ere
was a statistically signicant reduction in total cholesterol
and triglycerides as well as a numerical decrease in serum
glucose. In particular for triglycerides, this eect seems to
be less pronounced in rats exposed to AC ltered MS (−14%
for males, −16% for females) compared to those exposed to
control smoke (−39% for males, −57% for females).
Organ weight changes (Table 8) were qualitatively simi-
lar to those observed in previous MS inhalation studies,
although less pronounced. e main eect in absolute
organ weight compared to sham-exposed rats was the
MS-induced reduction in thymus weight, which was less
pronounced in AC ltered MS-exposed rats (–22% for males,
−20% for females) than in control MS-exposed rats of both
genders (−53% for males, −52% for females). A numerically
increased adrenal weight indicative of a stress response was
observed but obtained statistical signicance only for the
female rats, with no real dierentiation between test and
control MS-exposed groups. Also noted were changes in liver
weights and spleen weights which were less pronounced in
AC ltered MS-exposed rats.
Respiratory tract histopathological ndings occurring in
the anterior nose at level 1 included moderate reserve cell
hyperplasia and squamous metaplasia of the respiratory
epithelium at the lateral wall and on the nasoturbinates and
Table 6. Hematology measurements in rats exposed to MS from cigarettes containing AC lters compared to cellulose acetate ltered controls: set (b).
Parameter Unit Sham control (mean ± SD) 0 mg AC (mean ± SD) 180 mg AC (mean ± SD)
Male
Erythrocytes 1012/l 7.88 ± 0.12 8.24 ± 0.16 8.36 ± 0.13
Hematocrit l/l 0.421 ± 0.008 0.437 ± 0.006 0.445 ± 0.006
Hemoglobin g/l 151 ± 2 157 ± 2 158 ± 2
Mean corpuscular volume 53.4 ± 0.6 53.1 ± 0.6 53.2 ± 0.4
Mean corpuscular hemoglobin pg 19.2 ± 0.1 19.0 ± 0.2 18.9 ± 0.1
Mean corpuscular hemoglobin
concentration
g/l 359 ± 2 359 ± 2 354 ± 2
Leukocytes 109/l 6.47 ± 0.66 4.58 ± 0.41 5.36 ± 0.55
Neutrophils 109/l 0.96 ± 0.12 1.55 ± 0.18 1.11 ± 0.12
Eosinophils 109/l 0.07 ± 0.02 0.06 ± 0.02 0.05 ± 0.01
Lymphocytes 109/l 5.40 ± 0.62 2.89 ± 0.34 4.16 ± 0.43*
Monocytes 109/l 0.03 ± 0.01 0.07 ± 0.01 0.03 ± 0.01*
Platelets 109/l 867 ± 39 828 ± 42 802 ± 32
Female
Erythrocytes 1012/l 7.49 ± 0.08 7.82 ± 0.18 7.80 ± 0.13
Hematocrit l/l 0.399 ± 0.004 0.427 ± 0.008 0.430 ± 0.005
Hemoglobin g/l 145 ± 1 153 ± 3 155 ± 2
Mean corpuscular volume 53.3 ± 0.7 54.6 ± 0.7 55.2 ± 0.8
Mean corpuscular hemoglobin pg 19.4 ± 0.2 19.6 ± 0.3 19.8 ± 0.3
Mean corpuscular hemoglobin
concentration
g/l 365 ± 2 359 ± 3 359 ± 2
Leukocytes 109/l 5.20 ± 0.34 4.68 ± 0.43 5.58 ± 0.34
Neutrophils 109/l 0.58 ± 0.05 1.20 ± 0.15 0.99 ± 0.11
Eosinophils 109/l 0.06 ± 0.01 0.02 ± 0.01 0.06 ± 0.01
Lymphocytes 109/l 4.52 ± 0.35 3.40 ± 0.32 4.49 ± 0.30*
Monocytes 109/l 0.03 ± 0.01 0.04 ± 0.01 0.04 ± 0.01
Platelets 109/l 883 ± 23 851 ± 34 797 ± 53
Note: Bold print indicates statistically dierent from sham-exposed control (MS inhalation eect). Asterisk indicates signicant dierences from group
exposed to MS from cellulose acetate lter control (0 mg AC), p ≤ 0.05.
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Activated charcoal ltration of cigarette smoke 697
maxilloturbinates in rats of both genders exposed to MS of
the control cigarettes without AC ltration (Table 9). ese
changes were accompanied by goblet cell hyperplasia in
the respiratory epithelium of the septal region. In nose lev-
els 2–4, atrophy and squamous metaplasia of the olfactory
epithelium were observed. ese nasal eects were signi-
cantly less pronounced in nose level 1 or totally absent in
nose levels 2–4 in the rats exposed to MS of the test cigarettes
with AC ltration.
In the larynx, epithelial hyperplasia and squamous meta-
plasia were observed at various sites with variable incidence
and severity, with no relevant dierence noted between
the two types of cigarettes. e slight hyperplasia observed
in the pseudostratied epithelium at the upper medial
region of the vocal cords in the female rats was less pro-
nounced in the group exposed to AC ltered MS compared
to that exposed to control MS, whereas the slight squamous
metaplasia at this site was higher in the rats exposed to
the AC ltered MS. In a previous study, in which rats were
exposed to three concentrations of MS, a slight to moderate
squamous metaplasia was also described at this site, but this
eect was not dose-dependent (Terpstra et al., 2003). is
might indicate that the dierence observed in the current
study does not necessarily point to a higher potency of one
type of MS compared to the other, but might rather be part
of the scatter for this endpoint. In the male rats, only minor
MS-induced eects were seen at this site, with no dierence
between the two types of MS. At the vocal folds, hyperplasia
of the squamous epithelium was observed, which in both
genders was slightly less pronounced in the group exposed
to the AC ltered MS.
Eects in the larynx were also evaluated morphometri-
cally at the oor of the larynx and at the vocal cords. At both
sites, a clear MS eect could be seen (Table 10). e thickness
Table 7. Clinical chemistry measurements in rats exposed to MS from cigarettes containing AC lters compared to cellulose acetate ltered controls: set (b).
Parameter Unit Sham control (mean ± SD) 0 mg AC (mean ± SD) 180 mg AC (mean ± SD)
Male
Alanine aminotransferase U/l 60.5 ± 3.1 51.4 ± 2.7 42.9 ± 1.8*
Alkaline phosphatase U/l 169.3 ± 17.8 247.3 ± 37.2 178.6 ± 10.4
Aspartate aminotransferase U/l 65.7 ± 3.8 58.9 ± 2.4 56.3 ± 2.2
-Glutamyltransferase U/l 0.73 ± 0.14 0.75 ± 0.07 0.77 ± 0.09
Calcium mmol/l 2.71 ± 0.11 2.47 ± 0.04 2.45 ± 0.05
Chloride mmol/l 108.1 ± 1.0 105.6 ± 1.3 108.0 ± 1.6
Potassium mmol/l 5.72 ± 0.81 4.46 ± 0.19 4.40 ± 0.12
Sodium mmol/l 146.4 ± 0.9 145.6 ± 1.6 146.1 ± 2.0
Albumin % of protein 52.3 ± 1.2 54.9 ± 1.0 54.0 ± 0.8
Protein g/l 61.8 ± 1.3 56.2 ± 0.7 55.9 ± 0.9
Total bilirubin mol/l 2.54 ± 0.26 2.52 ± 0.12 2.34 ± 0.13
Urea mmol/l 6.55 ± 0.17 5.15 ± 0.25 5.78 ± 0.34
Creatinine mol/l 38.7 ± 3.1 33.8 ± 1.1 37.5 ± 1.5
Glucose mmol/l 15.4 ± 2.4 11.3 ± 0.5 11.1 ± 0.6
Total cholesterol mmol/l 2.05 ± 0.10 1.47 ± 0.05 1.52 ± 0.08
Triglycerides mmol/l 1.14 ± 0.07 0.69 ± 0.08 0.98 ± 0.09*
Inorganic phosphate mmol/l 2.07 ± 0.14 2.32 ± 0.11 2.41 ± 0.17
Female
Alanine aminotransferase U/l 52.2 ± 6.3 49.2 ± 2.3 40.5 ± 4.2
Alkaline phosphatase U/l 100.9 ± 7.5 161.1 ± 11.8 107.0 ± 7.9*
Aspartate aminotransferase U/l 68.1 ± 10.2 58.0 ± 2.0 58.6 ± 5.3
-Glutamyltransferase U/l 0.82 ± 0.13 0.83 ± 0.09 0.94 ± 0.09
Calcium mmol/l 2.76 ± 0.10 2.49 ± 0.07 2.44 ± 0.03
Chloride mmol/l 107.4 ± 1.1 105.1 ± 1.4 107.0 ± 0.6
Potassium mmol/l 4.93 ± 0.60 4.64 ± 0.34 3.96 ± 0.13
Sodium mmol/l 145.9 ± 0.9 144.1 ± 1.3 145.9 ± 0.6
Albumin % of protein 54.8 ± 1.1 54.7 ± 0.6 53.4 ± 1.0
Protein g/l 63.4 ± 1.9 55.5 ± 0.9 58.0 ± 0.9
Total bilirubin mol/l 2.11 ± 0.13 2.35 ± 0.11 2.41 ± 0.10
Urea mmol/l 6.57 ± 0.46 5.04 ± 0.40 4.91 ± 0.40
Creatinine mol/l 42.8 ± 3.2 38.7 ± 2.8 37.4 ± 0.9
Glucose mmol/l 16.0 ± 2.7 11.7 ± 2.1 10.8 ± 0.4
Total cholesterol mmol/l 2.45 ± 0.18 1.37 ± 0.09 1.75 ± 0.19
Triglycerides mmol/l 1.22 ± 0.17 0.53 ± 0.06 1.02 ± 0.09*
Inorganic phosphate mmol/l 1.62 ± 0.16 2.40 ± 0.13 2.03 ± 0.13
Note: Bold print indicates statistically dierent from sham-exposed control (MS inhalation eect). Asterisk indicates signicant dierences from group
exposed to MS from cellulose acetate lter control (0 mg AC), p ≤ 0.05.
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698 C. L. Gaworski et al.
of the epithelia at these sites numerically increased more in
groups exposed to AC ltered MS compared to those exposed
to control MS, a trend which was statistically signicant at
the vocal cords of the female rats (+20%).
e goblet cell hyperplasia observed in the respiratory
epithelia at the tracheal bifurcation and the main bronchus
was moderate to severe in the rats of both genders exposed
to the MS of the control cigarette, and was signicantly
reduced in severity and incidence in the groups exposed
to AC ltered MS. ere was only a very slight accumula-
tion of alveolar macrophages in the alveolar lumen of the
MS-exposed rats.
Set (c): eects of more intense smoking conditions
on AC-mediated changes in smoke composition
and in vitro toxicological activity
Test cigarettes with 180 mg of activated coconut shell char-
coal per cigarette were used to investigate the eciency of
the AC under the more intense MDPH smoking conditions.
Data for the AC-containing cigarette generated at ISO or
MDPH smoking conditions were normalized to those of the
Kentucky reference cigarette 2R4F smoked under the same
conditions and compared on a TPM relative basis.
MS composition
Per cigarette yields of the AC ltered cigarette for the gen-
eral Federal Trade Commission (FTC) parameters under
ISO conditions were 13.5 ± 0.1 mg TPM, 10.9 ± 0.1 mg
tar, 0.85 ± 0.02 mg nicotine, 1.73 ± 0.03 mg water, and
11.9 ± 0.3 mg carbon monoxide. ese parameters increased
under MDPH smoking conditions to 27.7 ± 0.3 mg TPM,
20.1 ± 0.5 mg tar, 1.38 ± 0.01 mg nicotine, 6.26 ± 0.16 mg water,
and 20.7 ± 0.4 mg carbon monoxide. e respective data for
the reference cigarette 2R4F under ISO conditions were
12.2 ± 0.2 mg TPM, 9.9 ± 0.2 mg tar, 0.91 ± 0.02 mg nicotine,
1.36 ± 0.03 mg water, and 14.2 ± 0.3 mg carbon monoxide.
ese values increased under MDPH smoking conditions to
30.5 ± 0.1 mg TPM, 22.3 ± 0.2 mg tar, 1.88 ± 0.01 mg nicotine,
6.31 ± 0.09 mg water and 26.0 ± 0.6 mg carbon monoxide.
A 70% decrease in the nitrosamine NNK and a 50% reduc-
tion in NNN yield compared to the reference cigarette when
smoked under MDPH conditions was noted and was attrib-
uted to the use of a specic blend of tobacco for the test ciga-
rette in this experiment.
In comparison between ISO and MDPH smoking condi-
tions, the yields for 1,3-butadiene, for example, increased
from 3.2 ± 0.2 to 14.4 ± 0.6 µg/cigarette (approximately 4.5-
fold, more than the 2.1-fold change in TPM) for the AC l-
tered cigarette and from 32.5 ± 0.6 to 57.8 ± 2.3 µg/cigarette
(approximately 1.8-fold, less than the 2.5-fold change for
TPM) for the reference cigarette 2R4F (Table 11). On an
equal TPM basis, this resulted in an approximately three-
fold higher relative yield of 1,3-butadiene at the more intense
MDPH smoking conditions compared to ISO smoking con-
ditions. Nevertheless, the activated AC in the test cigarettes
was still ecient in lowering 1,3-butadiene yield when
compared to the reference cigarette (e.g. −91% and −73%
for ISO and MDPH smoking conditions, respectively). e
eciency for acrolein removal by AC slightly decreased from
–96% (ISO) to –88% (MDPH). Similar trends with variable
Table 8. Organ weight measurements in rats exposed to MS from cigarettes containing AC lters compared to cellulose acetate ltered controls: set (b).
Parameter Unit
Weight (mean ± SD)
Sham control 0 mg AC 180 mg AC
Male
Lungs with larynx and trachea g 1.46 ± 0.05 1.41 ± 0.03 1.44 ± 0.04
Liver g 14.8 ± 0.4 10.9 ± 0.3 11.8 ± 0.5
Kidney (right) g 1.12 ± 0.05 0.98 ± 0.02 0.99 ± 0.04
Kidney (left) g 1.18 ± 0.04 0.97 ± 0.03 0.97 ± 0.03
Testis (right) g 1.72 ± 0.05 1.54 ± 0.04 1.52 ± 0.08
Testis (left) g 1.69 ± 0.05 1.54 ± 0.04 1.45 ± 0.07
Adrenal (right) g 0.027 ± 0.001 0.029 ± 0.001 0.028 ± 0.002
Adrenal (left) g 0.028 ± 0.001 0.031 ± 0.002 0.029 ± 0.002
Brain g 2.12 ± 0.04 2.00 ± 0.03 2.02 ± 0.02
ymus g 0.206 ± 0.020 0.096 ± 0.007 0.160 ± 0.016*
Spleen g 0.72 ± 0.04 0.44 ± 0.02 0.59 ± 0.05*
Female
Lungs with larynx and trachea g 1.18 ± 0.03 1.32 ± 0.04 1.32 ± 0.04
Liver g 9.9 ± 0.4 9.8 ± 0.3 9.7 ± 0.5
Kidney (right) g 0.78 ± 0.03 0.82 ± 0.02 0.76 ± 0.02*
Kidney (left) g 0.76 ± 0.03 0.79 ± 0.03 0.75 ± 0.02
Adrenal (right) g 0.029 ± 0.001 0.035 ± 0.002 0.035 ±0.002
Adrenal (left) g 0.032 ± 0.002 0.040 ± 0.002 0.037 ± 0.003
Brain g 1.97 ± 0.04 1.84 ± 0.03 1.91 ± 0.02*
ymus g 0.231 ± 0.014 0.108 ± 0.010 0.184 ± 0.017*
Spleen g 0.54 ± 0.03 0.42 ± 0.02 0.51 ± 0.03*
Note: Bold print indicates statistically dierent from sham-exposed control (MS inhalation eect). Asterisk indicates signicant dierences from group
exposed to MS from cellulose acetate lter control (0 mg AC), p ≤ 0.05.
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Activated charcoal ltration of cigarette smoke 699
magnitude were seen for other vapor phase constituents.
Vinyl chloride and 2-nitropropane were at the limit of quan-
tication for the AC ltered cigarette under ISO conditions
but could be quantied under MDPH smoking conditions.
e relative yields of particulate matter constituents, such as
the aromatic amines and the polycyclic aromatic hydrocar-
bons, were not aected by changing from one smoking con-
dition to another. Also, the relative nicotine yields were not
notably aected. us, under the conditions of this compar-
ative experiment, the major eect observed by using more
intense smoking conditions was less ecient reduction of
small vapor phase constituents, such as the aldehydes and
the aliphatic hydrocarbons.
In vitro cytotoxicity
For in vitro cytotoxicity of the TPM fraction, there was no
dierence in cytotoxic activity between the test and the ref-
erence cigarette at both smoking conditions (Table 12). For
the vapor phase fraction, the cytotoxicity for the AC ltered
cigarette relative to that of the reference cigarette was −95%
under ISO conditions and −90% under MDPH smoking con-
ditions. is slight reduction in eectiveness corresponds
to the slight reduction in relative trapping eciency of the
Table 9. Major histopathology results for rats exposed to MS from cigarettes containing AC lters compared to cellulose acetate ltered controls: set (b).
Finding
Male Female
Sham control
mean ± SD [n]
0 mg AC
mean ± SD [n]
180 mg AC
mean ± SD [n]
Sham control
mean ± SD [n]
0 mg AC
mean ± SD [n]
180 mg AC
mean ± SD [n]
Nose, level 1
Reserve cell hyperplasia 0.7 ± 0.4 [4/10] 3.6 ± 0.2 [10/10] 1.3 ± 0.3* [8/10] 0.0 ± 0.0 [0/10] 3.7 ± 0.2 [9/9] 1.1 ± 0.4* [6/10]
Goblet cell hyperplasia 0.4 ± 0.4 [1/10] 2.3 ± 0.4 [9/10] 0.8 ± 0.3* [5/10] 0.2 ± 0.1 [2/10] 1.8 ± 0.1 [9/9] 1.0 ± 0.2* [8/10]
Squamous metaplasia 0.3 ± 0.3 [1/10] 2.8 ± 0.1 [10/10] 0.3 ± 0.3* [1/10] 0.0 ± 0.0 [0/10] 1.9 ± 0.3 [9/9] 0.2 ± 0.1* [2/10]
Nose, level 2
Atrophy, olfactory epithelium 0.1 ± 0.1 [1/10] 2.9 ± 0.6 [8/10] 0.0 ± 0.0* [0/10] 0.0 ± 0.0 [0/10] 3.3 ± 0.4 [8/9] 0.0 ± 0.0 [0/10]
Squamous metaplasia 0.2 ± 0.2 [1/10] 2.0 ± 0.1 [10/10] 0.0 ± 0.0* [0/10] 0.0 ± 0.0 [0/10] 2.2 ± 0.4 [8/9] 0.0 ± 0.0 [0/10]
Nose, level 3
Atrophy, olfactory epithelium 0.0 ± 0.0 [0/10] 2.8 ± 0.6 [7/10] 0.0 ± 0.0* [0/10] 0.0 ± 0.0 [0/10] 3.9 ± 0.1 [3/9] 0.0 ± 0.0 [0/10]
Squamous metaplasia 0.2 ± 0.2 [1/10] 1.7 ± 0.2 [10/10] 0.0 ± 0.0* [0/10] 0.0 ± 0.0 [0/10] 2.4 ± 0.4 [8/9] 0.0 ± 0.0 [0/10]
Nose, level 4
Atrophy, olfactory epithelium 0.1 ± 0.1 [1/10] 1.5 ± 0.3 [9/10] 0.0 ± 0.0* [0/10] 0.0 ± 0.0 [0/10] 2.1 ± 0.3 [9/9] 0.0 ± 0.0 [0/10]
Squamous metaplasia 0.2 ± 0.2 [1/10] 1.7 ± 0.3 [9/10] 0.0 ± 0.0* [0/10] 0.0 ± 0.0 [0/10] 1.8 ± 0.3 [8/9] 0.0 ± 0.0 [0/10]
Larynx
Arytenoid projections, ventral depression
Hyperplasia 0.0 ± 0.0 [0/10] 0.8 ± 0.3 [5/9] 0.1 ± 0.1* [1/10] 0.0 ± 0.0 [0/9] 0.6 +/- 0.2 [5/9] 0.8 +/- 0.2 [6/9]
Squamous metaplasia 0.0 ± 0.0 [0/10] 0.2 ± 0.1 [2/9] 0.3 ± 0.2 [3/10] 0.0 ± 0.0 [0/9] 0.4 ± 0.3 [2/9] 0.4 ± 0.3 [2/9]
Floor of the larynx
Squamous metaplasia 0.0 ± 0.0 [0/10] 4.4 ± 0.3 [9/9] 4.3 ± 0.3 [10/10] 0.0 ± 0.0 [0/9] 4.0 ± 0.4 [9/9] 4.7 ± 0.2 [9/9]
Vocal cords, lower medial region
Squamous hyperplasia 0.0 ± 0.0 [0/10] 2.8 ± 0.1 [9/9] 2.7 ± 0.2 [10/10] 0.0 ± 0.0 [0/9] 2.8 ± 0.1 [9/9] 3.0 ± 0.0 [9/9]
Vocal cords, upper medial region
Hyperplasia 0.0 ± 0.0 [0/10] 0. 8 ± 0.1 [7/9] 0.8 ± 0.1 [8/10] 0.0 ± 0.0 [0/9] 0.9 ± 0.1 [8/9] 0.2 ± 0.1* [2/9]
Squamous metaplasia 0.0 ± 0.0 [0/10] 0.4 ± 0.3 [2/9] 0.5 ± 0.3 [2/10] 0.0 ± 0.0 [0/9] 0.2 ± 0.2 [1/9] 1.4 ± 0.4* [7/9]
Vocal folds
Squamous hyperplasia 0.3 ± 0.3 [1/10] 2.3 ± 0.4 [8/9] 1.6 ± 0.4 [7/10] 0.0 ± 0.0 [0/9] 2.7 ± 0.2 [9/9] 1.7 ± 0.3* [8/9]
Trachea, bifurcation
Goblet cell hyperplasia 0.2 ± 0.1 [2/10] 2.6 ± 0.4 [9/10] 0.9 ± 0.3* [6/10] 0.2 ± 0.2 [1/9] 2.0 ± 0.5 [6/8] 0.6 ± 0.3* [3/7]
Lung (left)
Goblet cell hyperplasia 1.0 ± 0.3 [6/10] 4.2 ± 0.4 [10/10] 0.9 ± 0.4* [5/10] 0.5 ± 0.4 [2/10] 4.4 ± 0.2 [9/9] 1.3 ± 0.4* [8/10]
Alveolar macrophages 0.0 ±0.0 [0/10] 0.3 ± 0.2 [3/10] 0.5 ± 0.2 [5/10] 0.0 ± 0.0 [0/10] 0.4 ± 0.2 [4/9] 0.7 ± 0.2 [7/10]
Note: Bold print indicates statistically dierent from sham-exposed control (MS inhalation eect). n = number observed/number examined. Asterisk
indicates signicant dierences from group exposed to MS from cellulose acetate lter control (0 mg AC), p ≤ 0.05.
Table 10. Larynx morphometry measurements in rats exposed to MS
from cigarettes containing AC lters compared to cellulose acetate lter
controls.
Parameter Unit
ickness (mean ± SD)
Sham control 0 mg AC 180 mg AC
Male
Floor of the larynx m11.6 ± 0.4 23.0 ± 0.9 27.3 ± 1.8
Vocal cords m18.9 ± 1.6 31.3 ± 1.5 32.8 ± 2.3
Female
Floor of the larynx m11.5 ± 0.5 25.5 ± 1.6 29.6 ± 1.5
Vocal cords m14.3 ± 0.6 31.7 ± 1.4 38.1 ± 1.6*
Note: Bold print indicates statistically dierent from sham-exposed
control (MS inhalation eect). Asterisk indicates signicant dierences
from group exposed to MS from cellulose acetate lter control (0 mg AC),
p ≤ 0.05.
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700 C. L. Gaworski et al.
trapping system for small molecular size vapor phase con-
stituents, such as acrolein.
Bacterial mutagenicity
For the bacterial mutagenicity of the TPM fraction, no repro-
ducible dierence was seen between the two cigarette types
under either smoking condition (data not shown). However,
there were two instances of a statistically signicantly higher
activity of the AC lter test cigarette compared with the
reference cigarette 2R4F: (1) under ISO smoking conditions,
one of two batches in tester strain TA98 with metabolic acti-
vation (+19% on a per mg TPM basis), and (2) under MDPH
smoking conditions, one of two batches in tester strain TA100
with metabolic activation (+46%). ere was no statistically
signicant dierence for the respective second batches in
these particular comparisons, nor was there a statistically
signicant dierence in the other tester strains under both
smoking conditions (data not shown).
Table 11. Smoke constituents analysis of MS collected under two smoking conditions from cigarettes containing AC lters compared to cellulose
acetate ltered controls: set (c).
ParameteraUnit/cigarette
ISO smoking conditions MDPH smoking conditions
2R4F
(mean ± SD)
180 mg AC
(mean ± SD) Ratio (%)b
2R4F
(mean ± SD)
180 mg AC
(mean ± SD) Ratio (%)b
Benzene g48.62 ± 0.99 1.40 ± 0.37 3 89.49 ± 2.46 4.79 ± 1.12 5
Toluene g82.39 ± 3.89 3.08 ± 1.42 4 174.44 ± 7.34 5.38 ± 1.88 3
Isoprene g358.0 ± 23.2 15.9 ± 1.9 4 658.3 ± 41.9 81.9 ± 12.1 12
Acrolein g66.33 ± 2.33 2.81 ± 0.41 4 128.14 ± 7.21 13.88 ± 1.01 11
Acrylonitrile g19.69 ± 0.46 0.87 ± 0.097 4 40.48 ± 1.52 3.785 ± 0.548 9
Propionaldehyde g56.98 ± 1.59 3.36 ± 0.39 6 103.66 ± 5.54 11.18 ± 0.68 11
Styrene g5.89 ± 0.40 0.48 ± c8 17.97 ± 0.94 0.797 ± 0.254 4
1,3-Butadiene g32.52 ± 1.20 3.16 ± 0.42 10 57.76 ± 4.69 14.35 ± 1.15 25
Acetaldehyde g685.5 ± 16.8 71.7 ± 6.6 10 1238.3 ± 66.7 298.4 ± 16.4 24
Hydrogen cyanide g103.9 ± 10.8 12.7 ± 1.5 12 239.2 ± 17.2 33.6 ± 2.4 14
Cadmium ng 38.0 ± 2.0 8.87 ± 0.46 23 93.3 ± 2.3 18.4 ± 0.4 20
NNK ng 129.1 ± 5.8 38.6 ± 1.3 30 239.5 ± 7.0 68.8 ± 6.8 29
N-nitrosonornicotine ng 148.4 ± 5.9 71.7 ± 3.2 48 283.7 ± 9.1 122.2 ± 8.8 43
Nitrogen oxides mg 311 ± 12 188 ± 4 60 539 ± 10 314 ± 9 58
Formaldehyde g19.3 ± 0.9 15.4 ± 0.8 80 41.1 ± 2.8 23.7 ± 0.6 58
Carbon monoxide mg 14.2 ± 0.5 11.9 ± 0.5 83 26.0 ± 1.1 20.7 ± 0.8 79
Phenol g9.09 ± 0.66 7.84 ± 0.49 86 16.80 ± 0.90 10.14 ± 0.18 60
Nicotine mg 0.91 ± 0.033 0.85 ± 0.031 94 1.878 ± 0.018 1.375 ± 0.013 73
Indeno(1,2,3-cd)pyrene ng 3.20 ± 0.09 3.17 ± 0.13 99 6.26 ± 0.43 5.33 ± 0.25 85
2-Naphthylamine ng 7.11 ± 0.79 7.11 ± 0.24 100 11.88 ± 0.75 11.66 ± 0.82 98
Lead ng 12.8 ± 1.6 12.8 ± 1.1 100 33.3 ± 1.2 28.5 ± 0.8 86
Benzo[a]pyrene ng 7.50 ± 0.36 7.81 ± 0.19 104 15.00 ± 0.95 13.17 ± 0.47 88
Catechol g45.3 ± 1.3 47.4 ± 1.8 105 88.5 ± 3.0 84.2 ± 2.9 95
4-Aminobiphenyl ng 1.36 ± 0.09 1.45 ± 0.08 106 2.77 ± 0.15 2.56 ± 0.20 92
Benzo[j]uoranthene ng 3.25 ± 0.10 3.47 ± 0.22 107 6.37 ± 0.84 5.97 ± 0.44 94
Benzo[b]uoranthene ng 5.39 ± 0.26 5.79 ± 0.22 107 11.41 ± 0.41 9.90 ± 0.27 87
Tar mg 9.9 ± 0.3 10.9 ± 0.1 110 22.3 ± 0.3 20.1 ± 0.9 90
Total particulate matter mg 12.2 ± 0.4 13.5 ± 0.1 111 30.5 ± 0.2 27.7 ± 0.6 91
o-Anisidine ng 2.21 ± 0.15 2.45 ± 0.17 111 4.47 ± 0.22 3.94 ± 0.20 88
Benzo[a]anthracene ng 11.93 ± 0.64 13.26 ± 1.91 111 24.88 ± 2.97 23.44 ± 3.17 94
Arsenic ng 3.40 ± 0.20 3.87 ± 0.31 113 7.73 ± 0.23 7.73 ± 0.23 100
Acetamide g6.81 ± 0.44 8.33 ± 0.24 122 17.59 ± 0.59 16.52 ± 0.95 94
o-Toluidine ng 55.9 ± 3.8 67.9 ± 2.6 122 107.2 ± 4.6 100.9 ± 3.1 94
Water mg 1.36 ± 0.05 1.73 ± 0.05 127 6.31 ± 0.17 6.26 ± 0.31 99
2-Nitropropane g15.92 ± 0.20 <3.9c— 25.86 ± 1.38 4.49 ± 0.18 17
Vinyl chloride ng 37.3 ± 0.6 <12.4 — 6.4 ± 6.1 26.3 ± 1.2 40
Benzo[k]uoranthene ng 2.40 ± c<2.40 — 3.49 ± 0.40 3.26 ± 0.51 93
Dibenzo[a,e]pyrene ng <0.60c<0.60c— 0.67c0.63 93
Note: e nitrosamines N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, and
N-nitrosopiperidine, the PAHs dibenz[a,h]anthracene, 5-methylchrysene, dibenzo[a,l]pyrene, dibenzo[a,i]pyrene, dibenzo[a,h]pyrene, and the elements
chromium and nickel were all below the quantitation limit for the assay.
an = 4 replicates with 4–20 cigarettes smoked per replicate.
bRatio (%) = (values for 0 mg AC/values for 2R4F) × 100.
cAt least one value below quantitation limit, median shown, SD not calculated.
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Activated charcoal ltration of cigarette smoke 701
Mammalian cell mutagenicity
e mouse lymphoma thymidine kinase assay, without and
with metabolic activation, was used to evaluate the TPM
fractions from control and AC ltered cigarettes. is assay
is performed in a mammalian cell line intended to comple-
ment the bacterial mutagenicity test. Under ISO smoking
conditions in the absence of the metabolic activation sys-
tem, the overall dierence between test and reference ciga-
rette was considered equivocal (Table 13). In the presence
of the metabolic activation system, there was no statistically
signicant dierence for the mutagenicity of either series
of batches between test and reference cigarette TPM. One
batch of each cigarette TPM was generated under MDPH
smoking conditions and used for this assay. Overall, the
mutagenic activity per mg TPM did not appreciably change
when using the more intense smoking conditions. ere was
also no dierence between the MS generated from the AC
ltered cigarette compared to that of the reference cigarette
without AC ltration, regardless of the absence or presence
of the metabolic activation system. us, AC ltration did not
measurably aect the mutagenic activity of the TPM fraction
in this mammalian genotoxicity assay.
Discussion
Consistent with previous reports of smoke constituent
analysis (Counts et al., 2005; Laugesen & Fowles, 2006;
Rees et al., 2007; Tokida et al., 1985), activated charcoal
in the lter design investigated here was ecient at trap-
ping many vapor phase constituents relative to the yields
of control cigarettes without AC ltration. When smoked
under ISO conditions, the addition of AC in the cigarette
lter reduced the presence of several vapor phase con-
stituents which have been identied individually by vari-
ous authoritative bodies and scientic investigations as
either irritants and/or carcinogens (e.g. acrolein, benzene,
cadmium, 1,3-butadiene, and formaldehyde). Even under
the more intense MDPH smoking conditions where the
cigarette lter is exposed to about 2–5 times as much MS
depending on the constituent and the cigarette construc-
tion (Counts et al., 2005), and there is a decrease in the
contact time because of an increased speed of the MS pass-
ing through the lter (Hammond et al., 2006), dierences
in vapor phase constituent yield between cigarette types
remained. e principal biological eects (i.e. reduced in
vitro cytotoxicity and irritant eects in the nasal epithelia
of the rats in the subchronic inhalation study) reasonably
matched the decreased yields of vapor phase constituents
reduced by activated charcoal.
While there was the expected reduction in vapor phase
constituents compared to control cigarettes, slight increases
in some particulate matter constituents were noted here and
have been inconsistently observed in other AC lter studies
we have conducted. While there is no readily apparent expla-
nation for increased particulate matter constituent yield in a
cigarette whose smoke is passed through an AC lter, a clue
to this eect may be found in the behavior of semivolatile
constituents found in smoke. Semivolatile constituents such
as nicotine and phenol have been reported to be trapped to
a certain extent by AC lters (Williamson et al., 1965). is
was also observed in the current study. is phenomenon
may hold true as well for other constituents not included on
the list of analyzed chemical constituents. To the extent they
are available in, or can redistribute fast enough to, the vapor
phase, these less volatile particulate matter constituents
may concentrate in the TPM and tar fractions and alter the
biological prole. is may explain some isolated ndings
of apparently greater toxicological activity associated with
the MS of the AC ltered compared to the cellulose acetate
ltered cigarettes when compared on a TPM basis (e.g. bac-
terial mutagenicity in TA98 after metabolic activation or the
more pronounced increase in epithelial thickness at the lar-
ynx). While these biological dierences disappeared when
compared on a cigarette basis, in some instances slightly
higher yields of particulate matter constituents even on a
Table 13. Mammalian cell mutagenicity of MS collected under two smoking conditions from cigarettes containing AC lters compared to the 2R4F
reference cigarette.
Metabolic
activation Smoking condition Batch
C3Ba (mg TPM/l) Statisticalb
2R4F (95% CL) 180 mg AC (95% CL) Per batch Overall
No ISO 1 38.7 (34.4–42.4) 43.4 (37.8–47.7) =
2 38.7 (35.0–42.0) 50.0 (46.2–53.1) * Equivocal
Yes ISO 1 169.5 (145.8– c) 157.3 (126.1–181.7) =
2 156.0 (141.1–169.4) 123.0 (100.8–144.2) = =
No MDPH 1 51.3 (48.7–53.4) 48.4 (44.6–52.5) =
Yes MDPH 1 147.2 (119.2–169.5) 128.2 (100.8–152.0) =
aC3B = concentration at which the mutant frequency increases three-fold beyond background.
bA dierence is determined if the 95% condence limits (CL) of the two cigarette types do not overlap.
cNo upper 95% CL for the C3B value could be determined because the lower condence limit of the dose–response curve did not exceed C3B.
Table 12. Cytotoxicity of TPM and vapor phase of MS collected under
two smoking conditions from cigarettes containing AC lters compared
to cellulose acetate ltered controls: set (c).
Smoke fraction
Smoking
condition
1/EC50 (ml/mg)
2R4F
(mean ± SD)
180 mg AC
(mean ± SD)
TPM ISO 8.56 ± 0.41 8.31 ± 0.46
MDPH 7.63 ± 0.18 8.09 ± 0.21
Vapor phase ISO 7.10 ± 0.48 0.36 ± 0.06*
MDPH 7.24 ± 0.19 0.76 ± 0.07*
Note: Asterisk indicates signicant dierences from group exposed to MS
from 2R4F, p ≤ 0.05.
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702 C. L. Gaworski et al.
per cigarette basis were observed. is cannot be explained
by the concentration eect discussed above, but may be
related to a given variability in the test cigarette construction
or chemical analysis.
In vitro cytotoxicity test results from the studies described
here indicated no apparent eect of AC ltration on the par-
ticulate phase cytotoxicity. However, a marked reduction of
the cytotoxicity of the vapor phase was noted, rendering it
almost impossible to determine an EC50 value even though
a wider dose range was used compared to that used for the
cellulose acetate ltered control cigarette. A similar trend
was previously reported for the whole MS of a cigarette con-
taining an AC lter with another design, an eect which was
associated with the reduction of vapor phase MS constitu-
ents (Bombick et al., 1997). Almost half of the cytotoxicity of
the MS vapor phase in this assay has recently been attributed
to acrolein and, to a lesser degree, to formaldehyde (Tewes
et al., 2003).
When compared on a per mg TPM basis, the particulate
matter phase of the AC ltered MS revealed a few instances
of greater genotoxic activity in the bacterial mutagenicity
assay compared to that of the cellulose acetate ltered con-
trol MS or the reference cigarette MS. As discussed above,
this may be either incidental or somehow related to a con-
centrating eect of the TPM fraction by increased trapping
of semivolatile constituents. No relevant dierences could
be detected between AC and cellulose acetate ltered ref-
erence MS for the genotoxic activity assessed in vitro by
the mouse lymphoma thymidine kinase assay. Previous
studies on another type of AC lter reported no AC-related
eect for the particulate phase in the bacterial mutagenic-
ity and a sister chromatid exchange assay (Bombick et al.,
1997). However, there was a reduced activity in the latter
assay when the whole smoke of the AC ltered cigarettes
was compared to that of the control cigarettes, suggesting
a possible AC ltration of MS constituents with genotoxic
potential.
Inhalation studies clearly utilize a more correct route
of exposure for cigarette smoke compared to routes and
smoke phases used for in vitro testing, and they carry con-
siderable weight in the evaluation of an aerosol such as
MS. However, the subchronic smoke exposure is generally
too brief to fully develop smoking-related diseases such
as lung cancer, emphysema, or atherosclerosis. Lifetime
MS inhalation studies have shown an increased incidence
of lung tumors in female rats (Mauderly et al., 2004). e
90-day subchronic inhalation study with MS of AC ltered
and cellulose acetate control cigarettes revealed biological
ndings consistent with those seen in previous smoke inha-
lation studies with a similar design (Terpstra et al., 2003;
Vanscheeuwijck et al., 2002). e eect on body weights seen
in rats exposed to smoke in this study is consistent with our
previous studies. While individual smoke constituents such
as nicotine (Chowdhury et al., 1989; Chowdhury, 1990) and
acrolein (Bouley et al., 1975; Feron et al., 1978) have been
implicated in eecting body weight, the apparent dieren-
tial between the sexes exposed to a whole smoke matrix is
not readily explainable. Exposure to cigarette smoke has
been reported to promote the release of neutrophils from
bone marrow into the peripheral blood (Terashima et al.,
1997, 1999), and in humans it is associated with increased
neutrophilic chemotactic activity (Anderson et al., 1991).
Contrasting MS-related changes in serum cholesterol and
triglyceride levels have been described in rats under vari-
ous exposure conditions and may be ascribed to changes
in the nutritional status in MS-exposed rats or changes in
their lipid metabolism (Jorge et al., 1995; Latha et al., 1988;
Maida & Howlett, 1990; Mikhail et al., 1979). ymic atro-
phy has been associated with exposure to irritant aldehydes
(Warholm et al., 1984).
e morphological changes observable in 90-day smoke
exposures including respiratory hyperplasia and metaplasia
may be taken as indicative of adaptive reversible responses
to the irritative properties of MS (Burger et al., 1989), which
upon long-term exposure may conceivably further develop
to pre-neoplastic eects. Goblet cell hyperplasia observed
in 90-day studies in the trachea and bronchus has been
considered an indication of chronic bronchitis (Harkema &
Wagner, 2002; White et al., 1986).
In the current study, the reserve cell and goblet cell
hyperplasias as well as the squamous metaplasia observed
in the respiratory epithelium of nasal level 1 were mark-
edly less pronounced in the rats exposed to AC ltered MS
compared to the corresponding eects in the rats exposed
to the MS of the control cigarettes. In addition, the atrophy
of the olfactory epithelium at nasal levels 2–4 seen in rats
exposed to control MS was totally absent in rats exposed
to AC ltered MS. Eects observable in the trachea and
the lungs have not been clearly assigned to particular
fractions of MS. In the current study, the major eect
was goblet cell hyperplasia in the respiratory epithelia of
the trachea and bronchus, which was markedly lower in
the rats exposed to AC ltered MS than in those exposed
to the control MS without AC ltration. A similar concord-
ance in decreased severity of ndings between the nose
and the trachea/bronchus was also described for rats
exposed to a less irritating modication of an electrically
heated cigarette smoking system, compared to its control
MS generated from cigarettes without this modication
(Moennikes et al., 2008).
ere was a trend toward increased epithelial thickness
in the rats exposed to AC ltered MS when the larynx epithe-
lium was evaluated by morphometric means (e.g. statistically
signicant in the vocal cords of the female rats). Previously,
it was shown that the isolated vapor phase of MS is mainly
involved in morphological changes in the nasal passages,
whereas the particulate phase is predominantly involved in
changes in the larynx (Coggins et al., 1980; Friedrichs et al.,
2006; Gaworski et al., 1998; Lam, 1980). Aerosol particles of
a given aerodynamic size or mass will impact the respira-
tory tract as a function of their aerodynamic characteristics
and velocity. Respiratory function determinations indicated
less pronounced respiratory depression in the AC ltered
MS-exposed female rats in this study. While the specic
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Activated charcoal ltration of cigarette smoke 703
reason for this is unknown, the nding suggests that females
may have been exposed to slightly higher MS doses of the AC
ltered cigarettes compared to those of the control cigarettes
without AC ltration. An increased peak inspiratory ow rate
was indeed found for the female rats in the current study,
which may result in more pronounced particle impaction
at the sensitive laryngeal epithelium. A similar laryngeal
response was observed in rats which were exposed to the
less irritating MS of an electrically heated cigarette smoking
system operated with modied cigarettes, compared to that
with unmodied cigarettes (Moennikes et al., 2008). As a
hypothesis, it may be suggested that the trend toward more
pronounced eects in the larynx of rats exposed to AC l-
tered MS compared to those observed in rats exposed to MS
without AC ltration resulted from altered inspiratory pat-
terns. e impact of this nding on human smokers of such
cigarettes is unknown.
e evidence obtained from the current series of studies
comparing MS of various AC ltered cigarette types to that
of matched control cigarettes or University of Kentucky ref-
erence cigarettes without AC lter suggests that the incor-
poration of this technology in conventional lit-end ciga-
rettes would not substantially increase the inherent toxicity
of cigarette MS, nor would it add new toxicity. e trend
toward increased epithelial thickness in the larynx of rats
subchronically exposed to AC ltered MS compared to the
eects seen in the rats exposed to MS generated without AC
ltration may be associated with decreased irritation in the
nasal passages, which may change the air ow and impact
of the MS particles at the laryngeal sites investigated. e
decrease of the morphological changes in the nose and the
lower respiratory tract as well as the decrease of the vapor
phase in vitro cytotoxicity associated with AC ltration is in
line with the ecient trapping of vapor phase constituents
demonstrated in the MS of cigarettes with AC lters. While it
is tempting to speculate on the overall eect of AC ltration
on the MS-related adult smoker cancer risk based solely on
MS compositional changes, any potential impact resulting
from a decreased presence of carcinogenic substances in
smoke remains vague as long as the constituent-specic
etiology of smoking-related cancer is not understood.
Furthermore, non-clinical studies, like those discussed
here, which may suggest reductions (or increases) in some
biological endpoints, only provide perspective about the
potential impact of AC cigarettes upon smoke toxicity.
Published epidemiological studies have failed to substanti-
ate reduced risk from smoking charcoal ltered cigarettes.
(Muscat et al., 2005; Stellman et al., 2001).
Acknowledgments
e authors are grateful to the sta at Philip Morris Research
Laboratories in Cologne, Germany, and Leuven, Belgium,
and the Illinois Institute of Technology Research Institute
in Chicago for the technical performance of the studies
described. ese studies were funded by Philip Morris
USA.
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