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Safety Assessment of Mainstream Smoke of Herbal Cigarette

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Owing to the increase in price of cigarettes in Korea, herbal cigarettes have received increasing attention as a non-smoking aid; however, its safety has hardly been studied. We analyzed some of the toxic components in the mainstream smoke of herbal cigarettes, performed a mutagenicity test on smoke condensates for safety assessment, and compared the results with the corresponding values of a general cigarette with the same tar content. Herbal cigarette "A" was smoked using automatic smoking machine under ISO conditions in a manner similar to general cigarette "T". The tar content measured was higher than that inscribed on the outside of a package. The mainstream smoke of herbal cigarette "A" did not contain detectable levels of tobacco-specific nitrosamines and nicotine. Carbon monoxide and benzo(α)pyrene contents in herbal cigarette "A" were higher than those in the general cigarette "T". The phenolic contents such as hydroquinone, resorcinol, and catechol in herbal cigarette "A" were higher than those in the general cigarette "T", but cresol contents in herbal cigarette "A" were lower than those in the general cigarette "T". The content of aromatic amines such as 4-aminobiphenyl in herbal cigarette "A" was higher than that in the general cigarette "T"; however, this difference was not statistically significant. On the other hand, 1-aminonaphthalene, 2-aminonaphthalene, and 3-aminobiphenyl contents in herbal cigarette "A" were lower than those in the general cigarette "T". The smoke condensates of herbal cigarette "A" exhibited a higher mutagenic potential than the condensates from the general cigarette "T" at the same concentration. We concluded that the mainstream smoke of herbal cigarette contains some toxic components, the smoke condensates of herbal cigarettes are mutagenic similar to general cigarette because of combustion products, and that the evaluation of the chemical and biological safety of all types of herbal cigarettes available on the market.
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41
Toxicol. Res.
Vol. 31, No. 1, pp. 41-48 (2015)
http://dx.doi.org/10.5487/TR.2015.31.1.041
plSSN: 1976-8257 eISSN: 2234-2753 Original Article
Open Access
Safety Assessment of Mainstream Smoke of Herbal Cigarette
Jong Ho Bak, Seung Min Lee and Heung Bin Lim
Department of Industrial Crop Science and Technology, Chungbuk National University, Cheongju, Korea
(Received February 3, 2015; Revised March 18, 2015; Accepted March 24, 2015)
Owing to the increase in price of cigarettes in Korea, herbal cigarettes have received increasing attention
as a non-smoking aid; however, its safety has hardly been studied. We analyzed some of the toxic compo-
nents in the mainstream smoke of herbal cigarettes, performed a mutagenicity test on smoke condensates
for safety assessment, and compared the results with the corresponding values of a general cigarette with
the same tar content. Herbal cigarette “A” was smoked using automatic smoking machine under ISO con-
ditions in a manner similar to general cigarette “T”. The tar content measured was higher than that
inscribed on the outside of a package. The mainstream smoke of herbal cigarette “A” did not contain
detectable levels of tobacco-specific nitrosamines and nicotine. Carbon monoxide and benzo(α)pyrene
contents in herbal cigarette “A” were higher than those in the general cigarette “T”. The phenolic contents
such as hydroquinone, resorcinol, and catechol in herbal cigarette “A” were higher than those in the gen-
eral cigarette “T”, but cresol contents in herbal cigarette “A” were lower than those in the general ciga-
rette “T”. The content of aromatic amines such as 4-aminobiphenyl in herbal cigarette “A” was higher than
that in the general cigarette “T”; however, this difference was not statistically significant. On the other
hand, 1-aminonaphthalene, 2-aminonaphthalene, and 3-aminobiphenyl contents in herbal cigarette “A”
were lower than those in the general cigarette “T”. The smoke condensates of herbal cigarette “A” exhib-
ited a higher mutagenic potential than the condensates from the general cigarette “T” at the same concen-
tration. We concluded that the mainstream smoke of herbal cigarette contains some toxic components, the
smoke condensates of herbal cigarettes are mutagenic similar to general cigarette because of combustion
products, and that the evaluation of the chemical and biological safety of all types of herbal cigarettes
available on the market.
Key words: Herbal cigarette, Safety, Assessment, Mainstream smoke, Toxic component, Mutagenicity test
INTRODUCTION
Cigarettes smoke is a complex mixture of chemical com-
ponents, many of which are known to be present in trace
concentrations (1). The various components are produced
by thermal decomposition and thermal synthesis reactions
occurring in the smoking process. There were 4,994 con-
firmed components in tobacco leaves and 5,311 confirmed
components in cigarettes smoke (2). Many components
identified in cigarette smoke were included in a list of haz-
ardous components in the International Agency for
Research on Cancer (IARC). All 9 components of them in
mainstream cigarettes smoke have an impact on the human
body and are classified as carcinogen (Group 1), as well as
the other 9 components of them are distinguished as likely
carcinogen to humans (Group 2A). They also contain 48
components that are possible carcinogen to humans (Group
2B) (3-5). The World Health Organization (WHO) recog-
nizes 18 components, Health Canada (HC) recognizes 44
components and the Food and Drug Administration (FDA)
of the USA recognizes 101 components as components
indispensable to control management. Generally tobacco
scientists regard 44 toxic components as Hoffmann list (6).
The quality of cigarettes smoke also is evaluated by biologi-
cal toxicity tests instead of content of harmful components.
Many biological toxicity tests such as genetic and cytotoxic
have been conducted using cigarettes condensates (7). Sal-
monella typhimurium TA98, TA100, TA102, TA1535, and
TA1537 have been used to repress the synthesis of specific
amino acids for genotoxicity testing of cigarettes smoke.
This method generated a morphological or functional muta-
Correspondence to: Heung Bin Lim, Department of Industrial Crop
Science and Technology, Chungbuk National University, Cheongju,
Chungbuk 362-763, Korea
E-mail: heungbin@chungbuk.ac.kr
This is an Open-Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0) which permits unrestricted
non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
42 J.H. Bak et al.
tion by impairing the DNA chromosomes that play an
important role in short-term retrieval methods for predict-
ing the carcinogenicity of the test substance (8).
The number of smokers who wish to stop smoking has
increased in recent years and smoking cessation education
has been conducted in many countries around the world.
However, it has been reported that success has occurred in
only 3~5% of smokers who wish to stop smoking during
one year; this low success rate is attributed to the lack of
will power and withdrawal symptoms due to nicotine
dependence (9). Many smokers who wished to stop smok-
ing use nicotine replacement therapy to help with with-
drawal symptoms (10). Non-smoking aids, such as nicotine
patches, nicotine gum, herbal cigarette and e-cigarette, are
used to help smoker stop smoking. Research suggests that
lots of smokers use herbal cigarette as a non-smoking aid in
Korea (11). The sales volume of electronic cigarette and
herbal cigarette according to G-market of on-line distribu-
tion industry in Korea have increased each 1,160%, 118%
in 2014, year-on-year. Also, sales volume of non-smoking
aids is expected on the increase because of raise of cigarette
price. Herbal cigarette contain herbs, instead of tobacco
leaves, as the raw material. Various herbal cigarette are sold
in the Korean commercial market. However, the safety and
chemical composition of herbal cigarette smoke have been
scarcely studied.
This study was performed to determine the contents of
some major toxic components in the mainstream smoke
of herbal cigarette “A” and to evaluate the mutagenicity
of smoke condensates and compare them with the corre-
sponding values of general cigarette “T” with the same tar
level.
MATERIALS AND METHODS
Materials. Herbal cigarette “A”, Artemisia (A), which
is most favorite in Korea market, and general cigarette (T)
with similar to tar content was used in this study. Selected
cigarette had a length of 84 mm and a circumferential of
8 mm. The tar and nicotine content of herbal cigarette “A”
was marked as 5.5 mg/cig and 0 mg/cig on the outside of
package, respectively. The tar and nicotine content of the
general cigarette “T” was indicated as 5.5 mg/cig and
0.6 mg/cig on the outside of package, respectively. All of
the samples were conditioned for 48 hrs at a temperature of
22 ± 1
o
C and a relative humidity of 60 ± 3% in the condi-
tioning room before smoking. The smoke was collected
according to the ISO 3402 method at a temperature of 22 ±
3
o
C and a relative humidity of 60 ± 5% (12). Automatic
smoking machines (ISO standardized products), RM 20
(Borgwaldt, Germany), SM 450 (Cerulean, UK) and SM
500 (Cerulean, UK), were used according to the ISO 3308
method under ISO standard smoking conditions, such as a
puff volume of 35.0 ± 0.3 mL, a puff duration of 60 ± 0.5
sec, a puff interval of 2.00 ± 0.02 sec and a butt length of
the filter tip paper plus 3 mm (13).
Measurement of the content of tar, nicotine and car-
bon monoxide (CO) in mainstream smoke. Isopropanol
(Merck, Germany) was used as an extraction solvent in
order to measure the content of tar, nicotine, and CO in the
mainstream smoke. Reference nicotine was purchased from
Sigma Aldrich co. (USA), and n-heptadecane (Sigma
Aldrich, USA) was used as an internal standard to analyze
the content of nicotine. Reference water was purchased
from Merck co. (Germany), and ethanol (Merck, Germany)
was used as an internal standard to measure the content of
water. The smoke was collected using RM 20 automatic
smoking machines (Borgwaldt, Germany), according to the
ISO 3308 method (13). Total particulate matter (TPM) was
calculated by measuring weight of the Cambridge filter pad
(CFP; Borgwaldt, Germany), in the cigarettes holder, before
and after smoking (14). The nicotine and water are extracted
by adding trapped CFP and extraction solution into the
flask, the supernatant was placed in a vial for analysis. The
nicotine and water content were analyzed using a gas chro-
matograph 6890N (GC; Agilent Technologies, USA) accord-
ing to the ISO 10315 and ISO 10362-1 method. According
to the ISO 8454 method, the CO content was automatically
measured by trapping gas in a gas bag that passed, through
the smoke trap in a RM 20 equipped with a CO analyzer
(Borgwaldt, Germany) (15-17).
Measurement of the content of aromatic amines in
mainstream smoke. The extraction solvent used to ana-
lyze the aromatic amines in the mainstream smoke was 5%
hydrochloric acid. The reference substances were 1-amino-
naphthalene, 2-aminonaphthalene, 3-aminobiphenyl, and 4-
aminobiphenyl (Sigma Aldrich, USA) and the internal stan-
dards were 2-aminonaphthalene-d7 and 4-aminobiphenyl-
d9 (CDN Isotopes, Canada) to analyze the content of aro-
matic amines. The smoke was collected using SM 500 auto-
matic smoking machines (Cerulean, UK) according to the
ISO 3308 method (13). The content of aromatic amines was
analyzed using GC/MS 5975 (Agilent Technologies, USA)
according to the HC T-102 method (18).
Measurement of the content of benzo[α]pyrene (B[α]P)
in mainstream smoke. Methanol (Merck, Germany) was
used as an extraction solvent to analyze the content of
B[α]P in the mainstream smoke. The reference substance
was B[α]P (Sigma Aldrich, USA) and the internal standard
was B[α]P-d12 (Sigma Aldrich, USA) to analyze the con-
tent of B[α]P. The smoke was collected using SM 500 auto-
matic smoking machines (Cerulean, UK) according to the
ISO 3308 method (13). The content of B[α]P was analyzed
using GC/MS 5975 (Agilent Technologies, USA) accord-
ing to the ISO 22634 method (19).
Safety Assessment of Mainstream Smoke of Herbal Cigarette 43
Measurement of the content of phenolic compounds
in mainstream smoke. The extraction solvent used to
analyze the phenolic compounds in the mainstream smoke
was 1% acetic acid solution in 100 mM ascorbic acid. The
reference substances were resorcinol, phenol, hydroqui-
none, catechol, o-cresol, p-cresol and m-cresol (Sigma
Aldrich, USA) to analyze the content of phenolic com-
pounds. The smoke was collected using SM 500 automatic
smoking machines (Cerulean, UK) according to the ISO
3308 method (13). The content of phenolic compounds was
analyzed using HPLC 1100 Series (Agilent Technologies,
USA) according to the HC T-114 method (20).
Measurement of the content of tobacco specific nitro-
samines (TSNAs) in mainstream smoke. The extraction
solvent used to analyze the TSNAs in the mainstream
smoke was 100 mM ammonium acetate. The reference sub-
stances were 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-
butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoa-
natabine (NAT) and N-nitrosoanabasine (NAB) (Sigma
Aldrich, USA) to analyze the content of TSNAs. The smoke
was collected using SM 450 automatic smoking machines
(Cerulean, UK) according to the ISO 3308 method (13).
The content of TSNAs was analyzed using HPLC/MS/MS
API 4000 (AB SCIEX, USA) according to the CORESTA
Recommended Method N
o
75 (21).
Mutagenicity test. Salmonella typhimurium TA98 strain
(frameshift mutation) was tested for genetic traits such as
histidine requirement, sensitivity to crystal violet (rfa),
ampicillin resistance and sensitivity to UV light (uvrB
mutation) prior to use (22). Dimethyl sulfoxide (DMSO),
agar, glucose, 2-aminoanthracene(2-AA), L-biotin, L-histi-
dine, citric acid monohydrate, potassium phosphate dibasic
anhydrous (K
2
HPO
4
), crystal violet, magnesium sulfate
(MgSO
4
·7H
2
O), sodium ammonium hydrogen phosphate
(NaNH
4
HPO
4
·4H
2
O), and ampicillin were purchased from
Sigma Co (St Louis, USA), S-9 (protein content : 23.6 mg/
mL) was purchased from Moltox Co (USA) and S-9 cofac-
tor was purchased from Oriental Yeast Co (Japan).
To determine the suitability of the strains verification
tests were performed in four ways. The histidine require-
ment confirmed whether S. Typhimurium TA98 grew in
minimal glucose agar (MGA), biotin, and histidine/biotin
plates after incubation at 37
o
C for 12 hr. For the uvrB muta-
tion, after inoculating the cultured S. Typhimurium TA98 on
the nutrient agar plate, half of the nutrient agar plate was
covered with aluminum foil and exposed to ultraviolet radi-
ation (15 W) at a distance of 33 cm for 8 sec. After 12 hr of
incubation, the portion exposed to ultraviolet radiation was
examined to determine whether any growth had occurred.
Cultured S. Typhimurium TA98 (100 μL) was added to the
top agar (2 mL), gently vortexed, and spread evenly on the
nutrient agar plate. A sterile filter paper disk was placed in
the center of the plate and 5 μL of crystal violet (2 mg/mL)
was dropped on the disk and allowed to absorb. After 12 hr
of incubation at 37
o
C, the diameter of the growth inhibition
ring around the disk was measured. R-factor plasmid con-
firmed whether S. Typhimurium TA98 grew on an ampicil-
lin plate after incubation at 37
o
C for 12 hr.
The smoke was collected using RM 20 automatic smok-
ing machines (Borgwaldt, Germany) according to the ISO
3308 method (13). The concentration of smoke condensate
in the mainstream smoke was extracted by adding trapped
smoke condensate in CFP with into the flask with DMSO
as a concentration of 4 mg/mL. After removing the bacteria
from the extracted smoke condensate using a 0.22 μm
syringe filter (Sartorius Stedim, Germany), the sample was
diluted to a concentration of 200 μg/mL in a micro-centri-
fuge tube (SPL, Korea). The mutagenicity test was con-
ducted using S. Typhimurium TA98, which is a modified
Ames test (22) First, 100 μL of the S. Typhimurium TA98
strain (1~2 × 10
9
cells/mL) was cultured in a sterile test tube
for 12 hr, and 100 μL of each sample was placed in a ster-
ile cap tube, to which 500 μL of S-9 mix was added for the
metabolic activation-requiring of the indirect mutagen test.
Then, 2 mL of 45
o
C top agar was added to each mixture,
gently stirred, and uniformly spread on minimal agar, fol-
lowed by 48 hr of incubation in the incubator at 37
o
C. The
positive controls 2-AA (1 μg/plate) served as the indirect
mutagen.
Statistical analysis. Data were analyzed by one-way
ANOVA followed by Dunnett’s multiple comparison test
(using SPSS version 14.0 statistical software). Differences
were considered statistically significant if P-values were
< 0.05 and < 0.01 by using ANOVA. The difference between
the herbal cigarette “A” and general cigarette “T” was
clearly distinguishable, and therefore, statistical signifi-
cance if comparisons between the herbal cigarette “A” and
general cigarette “T” has not been indicated.
RESULTS AND DISCUSSION
The content of tar, nicotine and CO in mainstream
smoke. Tar is TPM after deduction of its water and nico-
tine content in cigarette smoke and a black mixture of
hydrocarbons and free carbon obtained from a wide variety
of organic materials through destructive distillation. Nico-
tine is transferred directly into aerosol during smoking at a
temperature of approximately 220
o
C from a tobacco leaf.
CO is known to be generated by incomplete combustion at
a high temperature of approximately 700
o
C (23,24). Nico-
tine and CO are not confirmed in the evaluation of carcino-
genic levels of the IARC. The oral LD
50
of nicotine is
50 mg/kg. Nicotine is known to induce addiction, and CO
can cause fatal poisoning at a low concentration of approxi-
mately 667 μg/mL. CO poisoning causes coronary artery
44 J.H. Bak et al.
disease, such as angina pectoris or myocardial infarction
due to anoxia.
The contents of tar, nicotine, and CO in mainstream
smoke of herbal cigarette “A” and general cigarette “T” are
shown in Table 1. The indicated contents of tar, nicotine,
and CO on the certification of the reference cigarette were
9.5 mg/cig, 0.73 mg/cig and 12.0 mg/cig, respectively. The
measured contents of the tar, nicotine, and CO of the refer-
ence cigarette were 8.38 ± 0.07 mg/cig, 0.73 ± 0.02 mg/cig
and 12.53 ± 0.06 mg/cig, respectively. The indicated con-
tents of tar and nicotine on the package of general cigarette
“T” were 5.5 mg/cig and 0.60 mg/cig, respectively. The
measured contents of the tar, nicotine and CO of general
cigarette “T” were 6.02 ± 0.11 mg/cig, 0.57 ± 0.02 mg/cig
and 6.07 ± 0.25 mg/cig, respectively. They satisfied the tol-
erance range (package value ± 20%) of tar and nicotine.
Meanwhile, the indicated contents of the tar and nicotine on
the package of herbal cigarette “A” were 5.5 mg/cig and
0 mg/cig, respectively. The measured contents of tar of
herbal cigarette “A” was 7.45 ± 0.18 mg/cig, which exceed
the tolerance range (package value ± 20%) of tar in Korean
tobacco business law. However, nicotine of herbal cigarette
“A” was not detected. The content of CO of herbal ciga-
rette “A” was 12.30 ± 0.30 mg/cig.
We confirmed that tar and CO are present in the main-
stream smoke of herbal cigarette “A”, nicotine is not
detected, and the CO content of herbal cigarette is higher
than that of the general cigarette “T” with the same tar
level.
The content of aromatic amines in mainstream smoke.
Aromatic amines are formed under the pyrolysis conditions
underwent nitrogen at temperatures of approximately 650
o
C
produces during smoking (25). Aromatic amines are a com-
pound which bonded an amino group in the aromatic ring
and have high toxicity. 1-Aminonaphthalene is a compo-
nent of the herbicide and respiratory, and is not confirmed
in the carcinogenic of the IARC. The inhalation LC
50
(mouse) for 4 hr
of 1-aminonaphthalene is 0.056 mg/m
3
. 2-
Aminonaphthalene causes bladder cancer and is a carcino-
gen in group 1 in the carcinogenic of the IARC. The oral
LD
50
(mouse) of 2-aminonaphthalene is 727 mg/kg (3). 3-
Aminobiphenyle stimulates respiratory system, and is not
confirmed in the carcinogenic of the IARC. The oral LD
50
(mouse) of 3-aminobiphenyl is 331 mg/kg. 4-Aminobiphe-
nyl uses a dye and affects the nutritional metabolites and is
a carcinogen in group 1 in the carcinogenic of the IARC.
The oral LD
50
(mouse) of 4-aminobiphenyl is 500 mg/kg
(3).
The contents of aromatic amine in mainstream smoke of
herbal cigarette “A” and general cigarette “T” are shown in
Table 2. The measured content of 1-aminonaphthalene in
the reference cigarette was 16.67 ± 0.33 ng/cig similar to
the results of Chen and Moldoveanu (26). The measured
contents of 1-aminonaphthalene, 2-aminonaphthalene, 3-
aminobiphenyl and 4-aminobiphenyl among the aromatic
amine components of the general cigarette “T” were 12.60 ±
0.16 ng/cig, 7.48 ± 0.23 ng/cig, 1.52 ± 0.04 ng/cig and 0.89 ±
0.03 ng/cig, respectively. Meanwhile, the measured con-
tents of 1-aminonaphthalene, 2-aminonaphthalene, 3-ami-
nobiphenyl and 4-aminobiphenyl among the aromatic amine
components of herbal cigarette “A” were 8.23 ± 0.92 ng/
cig, 4.94 ± 0.36 ng/cig, 1.12 ± 0.14 ng/cig and 1.12 ± 0.14
ng/cig, respectively.
We confirmed that herbal cigarette “A” contains 4 toxic
aromatic amines in the mainstream smoke and their con-
tents were relatively low compared to the general cigarette
“T” with the same tar level.
The content of B[α]P in mainstream smoke. B[α]P
are formed under the pyrolysis conditions at temperatures
of approximately 860
o
C produces during smoking (27).
B[α]P is a compound which become metabolic activation in
Tab le 1. The contents of tar, nicotine and CO in mainstream
smoke
Compounds 3R4F “T” “A”
TPM (mg/cig) 10.39 ± 0.10 7.15 ± 0.14 8.33 ± 0.14
Tar (mg/cig) 08.38 ± 0.07 6.02 ± 0.11 7.45 ± 0.18
Nicotine (mg/cig) 00.73 ± 0.02 0.57 ± 0.02 N/d
CO (mg/cig) 12.53 ± 0.06 6.07 ± 0.25 12.30 ± 0.30
Values are expressed as mean ± S.E. (n = 3). Statistical analysis of
data was performed using ANOVA.
**
: p < 0.01 compared with control group.
3R4F: Reference cigarette. “T”: general cigarette with the contents
same tar. “A”: herbal cigarette made in Artemisia as raw material.
N/d: Not Detected.
Table 2. The contents of aromatic amines in mainstream smoke
Group Compound 3R4F “T” “A”
Aromatic amine
(ng/cig)
1-Aminonaphthalene 16.67 ± 0.33 12.60 ± 0.16 8.23 ± 0.92
2-Aminonaphthalene 11.97 ± 0.17 07.48 ± 0.23 4.94 ± 0.36
3-Aminobiphenyl 01.96 ± 0.05 01.52 ± 0.04 1.12 ± 0.14
4-Aminobiphenyl 01.95 ± 0.09 00.89 ± 0.03 1.00 ± 0.11
Values are expressed as mean ± S.D. (n = 3). Statistical analysis of data was analyzed using ANOVA.
**
: p < 0.01 compared with control group.
3R4F: reference cigarette. “T”: general cigarette with the contents same tar. “A”: herbal cigarette made in Artemisia as raw material.
Safety Assessment of Mainstream Smoke of Herbal Cigarette 45
vivo in poly-nuclear aromatic hydrocarbons and bind DNA
in the human body and can cause infertility by acting on
endocrine system, and is a carcinogen in group 1 in the car-
cinogenic of the IARC. The skin LD
50
(mouse) of B[α]P is
50 mg/kg (3).
The content of B[α]P in mainstream smoke of herbal cig-
arette “A” and general cigarette “T” is shown in Table 3.
The content of B[α]P in the reference cigarette was 14.11 ±
0.32 ng/cig similar to the results of Chen and Moldoveanu
(26). The measured content of B[α]P of the general ciga-
rette “T” was 2.29 ± 0.09 ng/cig. Meanwhile, the content of
B[α]P of herbal cigarette “A” was 2.77 ± 0.21 ng/cig.
We confirmed the presence of B[α]P in the mainstream
smoke of herbal cigarette “A” and its content is relatively
higher than the general cigarette “T” with the same tar
level.
Tab le 3. The contents of B[α]P in mainstream smoke
Compound 3R4F “T” “A”
B[α]P (ng/cig) 4.11 ± 0.32 2.29 ± 0.09 2.77 ± 0.21
Values are expressed as mean ± S.D. (n = 3). Statistical analysis of
data was analyzed using ANOVA.
*
: p < 0.05 compared with control group.
3R4F: reference cigarette. “T”: general cigarette with the contents
same tar. “A”: herbal cigarette made in Artemisia as raw material.
Table 4. The contents of phenolic compounds in mainstream smoke
Group Compound 3R4F “T” “A”
Phenolic compound
(μg/cig)
Hydroquinone 32.49 ± 1.61 27.03 ± 0.04 72.11 ± 3.48
Resorcinol 00.81 ± 0.04 00.79 ± 0.06 01.12 ± 0.01
Catechol 41.79 ± 1.25 39.78 ± 1.27 57.15 ± 2.85
Phenol 09.91 ± 0.70 14.86 ± 0.88 13.44 ± 0.55
M + p-Cresol 07.47 ± 0.54 08.34 ± 0.30 04.86 ± 0.13
O-Cresol 02.82 ± 0.16 03.00 ± 0.12 01.60 ± 0.07
Values are expressed as mean ± S.D. (n = 3). Statistical analysis of data was analyzed using ANOVA.
*
: p <0.05 and
**
: p < 0.01 compared with control group.
3R4F: reference cigarette. “T”: general cigarette with the contents same tar. “A”: herbal cigarette made in Artemisia as raw material.
Fig. 1. Chromatograms of phenolic compounds. A shows the chromatogram for the standard solution. B shows the chromatogram
for the herbal cigarette “A” of phenolic compounds.
The content of phenolic compounds in mainstream
smoke. Phenolic compounds are formed under the pyrol-
ysis conditions at temperatures of approximately 860
o
C pro-
duced during smoking (27). Phenolic compounds have a
peculiar smell because of a compound that combined
46 J.H. Bak et al.
hydroxyl group in the aromatic ring, and are toxic. Hydro-
quinone is a component affecting the central nervous sys-
tem inducing damage to the eyes and causing skin irritations,
and is not confirmed in the carcinogenic of the IARC. The
oral LD
50
(mouse) of hydroquinone is 317 mg/kg. Resorci-
nol cause hearing impairment and convulsions, phenol
causes damage to liver function by affecting the central ner-
vous system and are not confirmed in the carcinogenic of
the IARC. The oral LD
50
(mouse) of resorcinol and phenol
are 302 mg/kg and 301 mg/kg, respectively. Catechol is a
component affecting the respiratory system, is applied to an
oxidant of the dye owing to easy oxidation, and is a possi-
ble carcinogens in group 2B in the carcinogenic of the
IARC. The oral LD
50
(mouse) of catechol is 260 mg/kg (5).
O-cresol, m-cresol and p-cresol are components that cause
damage to the nose and lungs and are not confirmed in the
carcinogenic of the IARC. The oral LD
50
(mouse) of o-
cresol, m-cresol and p-cresol are 121 mg/kg, 242 mg/kg,
and 207 mg/kg, respectively.
The contents of phenolic compound in mainstream smoke
of herbal cigarette “A” and general cigarette “T” are shown
in Table 4. Fig. 1 shows the chromatograms for the stan-
dard solution and herbal cigarette “A”. The measured con-
tent of hydroquinone in the reference cigarette was 32.49 ±
1.61 μg/cig similar to the results of Chen and Moldoveanu
(26). The measured contents of hydroquinone, resorcinol,
catechol, phenol, m + p-Cresol, and o-Cresol among the
phenolic components of the general cigarette “T” were
27.03 ± 0.04 μg/cig, 0.79 ± 0.06 μg/cig, 39.78 ± 1.27 μg/cig,
14.86 ± 0.88 μg/cig, 8.34 ± 0.30 μg/cig, and 3.00 ± 0.12 μg/
cig, respectively. Meanwhile, the measured contents of
hydroquinone, resorcinol, catechol, phenol, m + p-cresol,
and o-cresol among the phenolic components of herbal cig-
arette “A” were 72.11 ± 3.48 μg/cig, 1.12 ± 0.01 μg/cig,
57.15 ± 2.85 μg/cig, 13.44 ± 0.55 μg/cig, 4.86 ± 0.13 μg/
cig, and 1.60 ± 0.07 μg/cig, respectively.
We confirmed that herbal cigarette “A” contains various
toxic phenolic compounds in the mainstream smoke, and
while the levels of some phenolic compounds such as
hydroquinone, resorcinol and catechol are relatively high,
the levels of other phenolic compounds such as o-cresol and
m + p-cresol are low compared to general cigarette “T”
with the same tar level.
The content of TSNAs in mainstream smoke. TSNAs
are formed by combining a secondary amine and nitrite
with a produced substance during drying process and the
process of machining of alkaloid such as nicotine, nornico-
tine, anatabine and anabasine in the tobacco leaf by the
pyrolysis (28). TSNAs are known as the causative agent of
oral perforation and liver, pancreas, lung and oral cancer.
NNN and NNK, in particular, and are a carcinogen in group
1 in the carcinogenic of the IARC (3). NAT and NAB are
not confirmed in the carcinogenic levels of the IARC. Com-
ponents are the most representative carcinogen in the ciga-
rettes smoke.
The contents of TSNA in mainstream smoke of herbal
cigarette “A” and general cigarette “T” are shown in Table
5. The measured content of NNN in the reference cigarette
was 116.22 ± 4.44 ng/cig similar to the results of Chen and
Moldoveanu (26). The measured contents of NNN, NAT,
NAB, and NNK among the TSNA components of the gen-
eral cigarette “T” were 45.78 ± 9.33 ng/cig, 41.62 ± 7.24 ng/
cig, 4.72 ± 0.33 ng/cig, and 16.38 ± 1.39 ng/cig, respectively.
Meanwhile, the TSNAs were not detected of herbal ciga-
rette “A”.
We confirmed that the toxic TSNAs are not detected in
the mainstream smoke of general cigarette “T” with the
same tar level.
Mutagenicity test. The histidine requirement test was
performed in a plate supplemented with histidine using S.
Typhimurium TA98 strains that were histidine auxotrophic
mutants of S. Typhimurium LT-2. T he Typhimurium TA98
strains grew on a histidine/biotin plate, but did not grow on
a MGA plate and a biotin plate (Fig. 2). UvrB mutation test
was also conducted to examine the sensitivity to UV test
must be not formed only in a portion UV of the radiation by
S. Typhimurium TA98 strains. In this test, the S. Typh-
imurium TA98 did not grow on the UV-exposed portions of
the plates, but grew on the portions that were not exposed to
UV light (Fig. 2). The rfa(Δ) mutation test was conducted
in which the mutation is indicated when the diameter of the
growth suppression ring around the filter paper disc was
greater than 14 mm. In the this test, the diameter of the
growth suppression ring that formed around the filter paper
disc was up to 16 mm (Fig. 2). The R-factor confirmation
test was performed using a strain that has the pKM101 plas-
mid, which is resistant to ampicillin. The results of the test
confirmed normal growth of the S. Typhimurium TA98
strain on an ampicillin plate (Fig. 2). Therefore, the suitabil-
ity of the S. Typhimurium strain TA98 for this study was
confirmed through genetic traits tests.
To establish the optimal concentration of the inducer for
mutagenic experiments prior to this test, the experiment was
Tab le 5. The contents of TSNAs in mainstream smoke
Group Compounds 3R4F “T” “A”
TSNAs
(ng/cig)
NNN 116.22 ± 4.44 45.78 ± 9.33 N/d
NAT 106.22 ± 6.81 41.62 ± 7.24 N/d
NAB 010.64 ± 0.63 04.72 ± 0.33 N/d
NNK 095.11 ± 8.57 16.38 ± 1.39 N/d
3R4F: reference cigarette. “T”: general cigarette with the contents
same tar. “A”: herbal cigarette made in Artemisia as raw material.
N/d: not detected.
NNN: N-Nitrosonornicotine. NAT: N-Nitrosoanatabine. NAB: N-
Nitrosoanabasine. NNK: 4-(N-Nitrosomethylamino)-1-(3-pyridyl)-1-
butanone.
Safety Assessment of Mainstream Smoke of Herbal Cigarette 47
repeated three times at each of the following concentrations
of smoke condensate; 50, 100, 200, 400, 800 and 1600 μg/
plate using a 3R4F of reference cigarette. The number of
revertant colonies of smoke condensate in mainstream
smoke of the reference cigarette is shown in Fig. 3. The
number of revertant colonies in the concentration of 50 μg/
plate was 215.0 ± 20.2, concentration of 100 μg/plate was
323.0 ± 12.5, concentration of 200 μg/plate was 639.7 ±
19.9 and concentration of 400 μg/plate was 789.7 ± 38.4.
Therefore, the number of revertants increased with the
increasing concentration until 400 μg/plate. However, the
number of revertant colonies at a concentration of 800 μg/
plate and of 1,600 μg/plate were decreased as 730.7 ± 15.6
and 454.7 ± 26.5 with the increasing concentration, respec-
tively. S. Typhimurim TA98 strain is assumed to not grow at
its toxicity when the concentration is more than 400 μg/
plate. Therefore, the smoke condensate concentration deter-
mined the 200 μg/plate for subsequent experiments.
The test results of mutagenicity of the smoke condensate
of herbal cigarette “A” and general cigarette “T” in main-
stream smoke are shown in Table 6. In the indirect muta-
gen test (+S-9 mix), the mutagenicity was 46.67 ± 3.21
from the number of spontaneous revertant colonies in the
negative control group (DMSO) and mutagenicity was
1238.00 ± 95.85 in the positive control group (2-AA).
When 200 μg/plate of smoke condensate was added, muta-
genicity of general cigarette “T” was 518.7 ± 30.0. Mean-
while, mutagenicity of herbal cigarette “A” was 539.7 ± 29.7.
We confirmed that the smoke condensates of herbal ciga-
rette “A” could induce mutagenicity, and its degree is higher
than the general cigarette “T” with the same tar level.
We concluded that some toxic components also exist in
the mainstream smoke of herbal cigarette “A” in large
amounts similar to general cigarette. Although tobacco-spe-
Fig. 2. Genetic traits test of S. Typhimurium TA98. Genetic traits test was indicated activation of S. Typhimurium TA98. MGA plate; Mini-
mal glucose agar plate, Biotine plate; 0.5 mM Biotine solution (122.1 μg/mL), Histidine/Biotine Plate; 0.5 mM Biotine solution (122.1 μg/
mL) and Histidine solution (5 mg/mL), UvrB mutation plate; Exposed to ultraviolet radiation (15 W) at a distance of 33 cm for 8 sec,
rfa(Δ) mutation plate; Dropped on 5 μl of crystal violet solution (2 mg/mL), Ampicillin plate; Ampicillin solution (10 mg/mL of 0.02
sodium hydroxide).
Fig. 3. Change of revertant colony number with smoke con-
densate concentration in mutagenicity test. The S. Typhimurium
strains TA98 were treated with the indicated concentration of
3R4F smoke condensate for 48 hr. Values are expressed as a
mean ± S.D (N = 3).
48 J.H. Bak et al.
cific components such as nicotine and TSNA were not
detected, the smoke condensates of herbal cigarette “A”
have mutagenic potential similar to general cigarette.
Hence, the chemical and biological safety of herbal ciga-
rettes should be assessed periodically.
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Table 6. The mutagenicity test of smoke condensate
Treatment
(+S-9 mix)
Concentration
(μg/plate)
Revertants/plate
Spontaneous 046.67 ± 3.21
Control (2-AA) 001 1238.00 ± 95.85
Smoke condensate
“T” 200 0518.67 ± 29.96
“A” 200 0539.67 ± 29.70
Values are expressed as mean ± S.D. (n = 3). Statistical analysis of
data was analyzed using ANOVA.
T”: general cigarette with the contents same tar. “A”: herbal ciga-
rette made in Artemisia as raw material.
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