Regulatory Toxicology and Pharmacology 76 (2016) 113e120

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Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Tobacco-specific N-nitrosamines NNN and NNK levels in cigarette

brands between 2000 and 2014
I. Gunduz a, *, A. Kondylis a, G. Jaccard b, J.-M. Renaud a, R. Hofer a, L. Ruffieux c, F. Gadani a
a
Philip Morris Products SA, Product Development, Rue des Usines 56, 2000 Neucha^tel, Switzerland
b
Philip Morris International Management SA, Product and Process Quality, Quai Jeanrenaud 56, 2000 Neucha^tel, Switzerland
c
Philip Morris International Management SA, Leaf Agricultural Programs, Avenue de Rhodanie 50, 1001 Lausanne, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: The evolution of the levels of tobacco-specific N-nitrosamines (TSNA), N-nitrosonornicotine (NNN) and
Received 9 December 2015 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mainstream (MS) cigarette smoke is inves-
Received in revised form tigated based on smoke and tobacco chemistry data of cigarette brands sold by Philip Morris Interna-
7 January 2016
tional (PMI) between 2000 and 2014. A total of 315 cigarette samples representing a wide range of
Accepted 20 January 2016
Available online 22 January 2016
product and design characteristics manufactured by PMI between 2008 and 2014 were analyzed and
compared to a previously published dataset of PMI brands manufactured in 2000. The data indicate that
there is a substantial reduction of NNN and NNK levels in tobacco fillers and MS cigarette smoke per mg
Keywords:
TSNA
of tar and per mg of nicotine using Health Canada Intense (HCI) machine-smoking regime. This observed
NNN reduction in NNN and NNK levels in MS cigarette smoke is also supported by the downward trend
NNK observed on NNN and NNK levels in USA flue-cured Virginia and Burley tobacco lots from 2000 to 2014
Tobacco crops, reflecting effectiveness of measures taken on curing and agricultural practices designed to
Cigarette smoke minimize TSNA formation in tobacco.
Smoke transfer © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
The tobacco specific nitrosamines (TSNA) 4-(methylni-
trosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitro-
sonornicotine (NNN) are classified as group 1 carcinogens by the
International Agency for Research on Cancer (IARC, 2007). Both
compounds have been identified in the list of harmful and poten-
tially harmful constituents (HPHCs) in tobacco products and to-
bacco smoke by the US Food and Drug Administration (Food and
Drug Administration, 2012). They have also been prioritized by
others for regulatory purposes (World Health Organization, 2008;
Talhout et al., 2011; Burns et al., 2008; Xie et al., 2011).
TSNAs are formed by nitrosation of alkaloids present in the to-
bacco plant (Fischer et al., 1989). It is generally accepted that NNN is
formed via nitrosation of nornicotine, a secondary alkaloid gener-
ated directly from nicotine through activity of the enzyme nicotine
demethylase. NNK is formed from nitrosation of nicotine or its
oxidized products (Fischer et al., 1989; Piade  et al., 2013) and is
found in two forms in tobacco, a free (soluble) and a matrix-bound
* Corresponding author.
E-mail address: irfan.gunduz@pmi.com (I. Gunduz).
(insoluble) form (Lang and Vuarnoz, 2015). The matrix-bound form
of NNK results from incorporation of nicotine or its oxidized
product, pseudooxynicotine during lignin polymerization. On
average, 77% and 53% of the total NNK were matrix-bound in air-
cured (Burley type) and flue-cured tobaccos, respectively (Lang
and Vuarnoz, 2015).
A typical American blended cigarette cut-filler contains three
types of tobaccos including flue-cured Virginia, Burley and Oriental.
Burley and flue-cured Virginia tobaccos are the main source of NNN
and NNK in a typical American blend and as a result in cigarette
smoke. Typically NNN is predominant in Burley tobaccos, while
NNK is a major TSNA in flue-cured Virginia tobaccos (d'Andres et al.,
2003). Oriental tobaccos are grown in poor soils with little if any
added nitrogen fertilizer, are sun-cured and typically contain very
low nitrate (Leffingwell, 1999), leading to very low levels of TSNA.
Burley tobaccos are air-cured and are typically grown with higher
nitrogen fertilization than flue-cured Virginia and Oriental
tobaccos. Moreover, Burley tobaccos contain also higher amounts of
nornicotine, a precursor of NNN, and nitrate, a precursor of nitrite, a
nitrosation agent. While in green tobacco leafs TSNA occur only in
trace amounts (Djordjevic et al., 1987), higher concentrations are
formed during senescence and air curing when secondary alkaloids

http://dx.doi.org/10.1016/j.yrtph.2016.01.012
0273-2300/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
114 I. Gunduz et al. / Regulatory Toxicology and Pharmacology 76 (2016) 113e120
are nitrosated by nitrite formed microbially from nitrate (Burton
et al., 1989, 1994). Flue-cured Virginia tobaccos contain lower
amount of nitrate and are cured in a controlled environment which
takes 5e7 days. Formation of TSNA in flue-cured tobacco is pre-
dominantly through the reaction of alkaloids in leaf with nitrogen
oxides present in combustion gases formed during the curing
process, if the flue-curing process is performed with flue gases from
an open flame (Nestor et al., 2003).
During the past several decades, a number of measures, focusing
on precursors' reduction, have been taken to decrease both NNN
and NNK in flue-cured Virginia and Burley tobaccos. These mea-
sures include:
1 Barn conversion - An industry wide initiative to retrofit direct-
fired flue-cured barns with indirect-fired heating started
around 2000 (Reed, 2009) after the discovery that nitrogen
oxides preformed with flue gases from an open flame are the
cause of high TSNA in flue-cured Virginia tobaccos (Nestor et al.,
2003). As a result more than 30,000 barns in the USA have been
retrofitted from direct-fired to the heat-exchanger system with
some of the expense reimbursed by an industry supported cost
share program (Reed, 2009). Research conducted after barn
conversion indicates that the levels of TSNA in flue-cured
tobaccos in USA (Nestor et al., 2003) and in Canada, where a
similar industry-wide effort has been undertaken, have been
reduced (Rickert et al., 2008).
2 Use of Burley low-converter seed - Burley tobaccos contain
considerably higher levels of nornicotine than flue-cured Vir-
ginia tobaccos (Jack et al., 2007) due to an increased propensity
of conversion of nicotine to nornicotine from one generation to
another in Burley tobaccos (Mann et al., 1964; Jack et al., 2007;
Lewis et al., 2010). The frequency of the conversion may be as
high as 20% per generation in some Burley cultivars (Siminszky
et al., 2005).
A screening process was developed to reduce levels of norni-
cotine present in a given Burley tobacco variety through
selecting plants having less than 3% nornicotine relative to
nicotine content prior to seed harvest (Miller et al., 2004; Jack
et al., 2007). This screening process is utilized to release exist-
ing Burley varieties as low-converter (LC) varieties since 2005 in
USA (Miller, 2005) and other countries and should result in
lower NNN levels in tobaccos, as nornicotine is a known pre-
cursor of NNN (Brunnemann et al., 1996). More recent research
on the mechanism of nornicotine formation led to the identifi-
cation of three nicotine demethylase genes, CYP82E4
(Siminszky et al., 2005), CYP82E5 (Dewey et al., 2007) and
CYP82E10 (Lewis et al., 2010) that facilitate conversion of
nicotine to nornicotine. Tobacco varieties with mutant version
of these three genes show stable levels of ~0.5% nornicotine
relative to nicotine (Lewis et al., 2010). Major Burley commercial
varieties containing three non-functional versions of these
genes were approved in 2013 and 2014 in the Regional Mini-
mum Standards program conducted cooperatively by Univer-
sities in Georgia, South Carolina, North Carolina and Virginia.
3 Adoption of good agricultural practices recommended by the
University of Kentucky, North Carolina State University, Virginia
Polytechnic Institute and State University, and University of
Tennessee (University of Kentucky College of Agriculture,
University of Tennessee Institute of Agriculture, North Carolina
State University and Virginia Tech, 2015) is targeted to: a-
Reduce nitrate pool via recommended nitrogen usage as corre-
lation between increased nitrogen fertilization with increased
TSNA in tobacco is widely recognized (Chamberlain and
Chortyk, 1992). b- Minimize nitrate reduction to nitrite by
reducing humidity and speeding up curing process in Burley
tobacco through barn ventilation. Higher humidity and tem-
peratures during air curing leads to higher levels of nitrite and
TSNA in air-cured tobaccos (Burton et al., 1989). Burley tobacco
leaves cured in well ventilated curing barns generally contain
less amount of TSNA than leaves cured in a conventional curing
barn (De Roton et al., 2005).
In recent studies, it has been demonstrated that NNN and NNK
levels have decreased over a 35-year time span in the tobacco fillers
and mainstream smoke of cigarettes sold in USA (Appleton et al.,
2013) and that the exposure to TSNA, as measured by NNAL uri-
nary excretion data, was reduced as well over a 18-year period
(Appleton et al., 2014). Those results were in contradiction with the
claim, based on the analysis of a limited number of cigarettes sold
in USA, that no change had been observed over the last 3 decades
for TSNA levels (Stepanov et al., 2011).
In this work, we have compiled and analyzed published (Counts
et al., 2005) and recent internal data to assess the effectiveness of
the measures taken on curing and agricultural practices designed to
reduce TSNA formation in tobacco. The former data result from
cigarette brands sampled in 2000, while the latter are obtained
from benchmark surveys conducted in 2008, 2010, 2012 and 2014.
Changes in NNN and NNK levels were investigated in tobacco filler
and mainstream smoke. In addition, TSNA values from Burley and
flue-cured Virginia tobacco lots bought by PMI in the USA between
2000 and 2014 were also investigated to confirm whether good
agricultural practices designed to minimize TSNA formation are
effective.
2. Materials and methods
2.1. Cigarettes
The brands under study were commercialized American blend
cigarettes manufactured and sold by PMI during the period of
2008e2014. The data from these brands are compared to published
data containing PMI brands (Counts et al., 2005). The latter will be
referred to as PMI 2000 benchmark study here after. PMI 2000
benchmark study covered 48 brands including various cigarette
design features. The cigarettes were sampled at fourteen produc-
tion facilities in late 1999. In this study data from 44 American
blended cigarettes were kept for comparison, while the remaining
non-American blended cigarettes were excluded from the analysis.
The PMI internal benchmark surveys were conducted in 2008,
2010, 2012, and 2014. Excluding non-American blended cigarettes,
88 PMI brands were sampled from 11 markets in 2008, 126 brands
from 26 markets in 2010, 76 brands from 19 markets in 2012, and
25 brands from 11 markets in 2014. The selection in each individual
market was performed to reflect as much as possible the markets in
terms of cigarette designs and formats.
2.2. Mainstream smoke and tobacco filler analyses
The selected brands were analyzed for TSNA in both tobacco
filler and mainstream smoke using the same analytical methods
and laboratory that was reported in PMI 2000 benchmark study.
The chemical analyses were conducted under contract to PMI by
Labstat International ULC (Kitchener, Ont. Canada), an ISO 17025
accredited laboratory. TSNA in tobacco fillers and mainstream
smoke were analyzed by High Performance Liquid Chromatography
with tandem mass spectrometry detection (LC-MS/MS) according
to the Labstat internal methods T-309 and T-111 respectively; three
replicates per sample were conducted. After conditioning according
to the International Organization for Standardization standard
 I. Gunduz et al. / Regulatory Toxicology and Pharmacology 76 (2016) 113e120 115

method ISO-3402 (International Organization for Standardization, Table 1

1999), cigarettes were machine-smoked under Health Canada Mean values (AVG) and their associated standard error (SE) for NNN and NNK in
tobacco filler and mainstream cigarette smoke (MS). Numbers in the parenthesis
Intense machine-smoking conditions (Health-Canada, 1999). Tar, indicate number of sampled brands.
nicotine and carbon monoxide (CO) in mainstream smoke were
analyzed according to method T-115 (Health-Canada, 1999). Eight Study Statisitc Tobacco filler MS [ng/mg SN] MS [ng/mg
[ng/g] tar]
replicates per sample were performed. The NNN and NNK yields in
smoke were normalized, either to smoke nicotine (SN) yield or to NNN NNK NNN NNK NNN NNK

tar yield, to be able to correct for different nicotine/tar yields dis- 2000 (44)* AVG 2236.5 808.1 117.54 72.30 7.61 4.68
tribution in the yearly survey samplings. SE 142.7 50.3 6.57 3.31 0.4 0.2
2008 (88) AVG 1471.9 313.5 94.96 56.46 6.1 3.6
SE 70.6 14.8 4.30 3.03 0.3 0.2
2.3. Tobacco lots analyses 2010 (126) AVG 1352.7 335.1 79.45 56.27 5.1 3.6
SE 39.4 11.2 2.39 2.04 0.1 0.1
All tobacco lot samples considered in the present study were 2012 (76) AVG 1598.8 319.7 95.99 60.03 5.6 3.5
flue-cured Virginia and Burley tobacco lots grown in the USA be- SE 55.1 9.3 3.82 2.13 0.2 0.1
2014 (25) AVG 985.1 334.2 74.86 52.17 4.8 3.3
tween 2000 and 2014 and purchased by PMI. A tobacco lot is
SE 79.7 25.5 4.96 3.34 0.3 0.2
composed of tobaccos of same type from the same origin, the same
*
Data extracted from the PMI 2000 benchmark study. SN e Smoke nicotine.
crop year, and same plant stalk positions. Lamina samples from
different tobacco lots were sampled after threshing in PMI ware-
houses (crops 2000e2008) or at raw tobacco processor location
The results in Tables 1 and 2 show statistically significant
(2009 crop and later). The analyses of TSNA in single grade tobacco
reduction of the NNN and NNK levels both in mainstream cigarette
lots were performed in four different laboratories using LC-MS/MS
smoke and in tobacco filler in recent years as compared to 2000.
according to their internal methods.
The observed reductions vary from 17 to 37% in mainstream smoke
and 29e61% in tobacco filler.
2.4. Data treatment and analysis
Mean values of NNN in tobacco filler of sampled products were
reduced from 2237 ng/g in 2000 to 1472 ng/g in 2008. In consec-
Mainstream smoke constituents data have been received from
utive years after 2008 average NNN levels fluctuated between
Labstat on a per cigarette basis. Smoke nicotine and tar yields have
minimum 985 to maximum 1599 ng/g tobacco filler (Table 1). Mean
been used in order to adjust the NNN and the NNK yields per
values of NNN per mg of smoke nicotine (SN) followed similar
milligram of smoke nicotine or per milligram tar, respectively. The
trends and were reduced from 118 ng/mg SN to 95 ng/mg SN in
PMI 2000 benchmark data have been extracted from published
2008. After 2008 average NNN levels using the HCI machine-
data (Counts et al., 2005). For both mainstream smoke and tobacco
smoking regimen, fluctuated between a minimum average of
filler data interest focused on comparing the TSNA levels of the
75 ng/mg SN and a maximum average of 96 ng/mg SN.
recent PMI data to the PMI 2000 benchmark data. This has been
Mean values of NNK in tobacco filler of sampled products were
done by estimating the mean difference between the TSNA yields in
reduced from an average 808 ng/g tobacco filler in 2000 to 314 ng/g
recent studies and year 2000, and assessing whether the observed
tobacco filler in 2008. In consecutive years NNK levels in tobacco
difference is statistically significant. Given the multiplicity of the
filler remained quite stable with a minimum average of 320 to
testing problem (2008, 2010, 2012, and 2014 yields are compared to
maximum average of 335 ng/g tobacco (Table 1). Average NNK
2000 yields), an over-all 5% error rate using the Dunnett multiple
values in mainstream cigarette smoke using HCI smoking regimen
testing correction was applied (Miller, 1981). Confidence intervals
were reduced from 72 to 56 ng/mg SN in 2008. In consecutive years
(CIs) were computed and reported for which the over-all (simul-
they fluctuated between 52 and 60 ng/mg SN.
taneous) confidence level was 95%; their individual confidence
Similar reductions in mainstream smoke were also observed
level is 98.37%. For USA tobacco lots, data are available for a large
when both NNN and NNK were normalized per mg of tar (Table 1).
period (spanning the range from 2000 to 2014) and trends for
The simultaneous 95% CIs on the reduction of NNN and NNK levels
reduction are tested by means of linear regression to the average
in tobacco filler and in mainstream smoke are also illustrated in
TSNA yields across time. Data are analyzed in the appropriate scale
Fig. 1. The CIs are plotted in relative terms after dividing the esti-
in order to meet linearity and apply statistical tests for testing the
mated difference and the associated lower and upper limits by the
linear regression slope. Statistical testing was performed on the 5%
average reference yield in 2000. Fig. 1 depicts the global reduction
significant level.
of both NNN and NNK levels of all recent PMI data compared to
2000, and the higher reduction in tobacco filler compared to
3. Results
mainstream smoke.
Tobacco lot results across the time span 2000e2014 are given in
Mean NNN and NNK values for tobacco filler and mainstream
Table 3 and Table 4. Results include year averages with their asso-
smoke (normalized per mg of tar and nicotine) measured in ciga-
ciated SE, as well as number of lots analyzed and number of lots
rette samples from the PMI 2000 benchmark study and the PMI
with TSNA above detection limits. The tobacco lot results are
benchmark surveys conducted in 2008, 2010, 2012, and 2014 are
illustrated in Fig. 2 and Fig. 3. The average NNN and NNK values are
presented in Table 1. The variation of the means as measured by
depicted together with their associated 95% confidence intervals.
their standard error (SE) is also reported in Table 1. Results on the
The analysis of individual flue-cured Virginia tobacco lots pur-
statistical assessment of the comparisons for NNN and NNK in to-
chased in USA shows that both NNN and NNK levels substantially
bacco filler and mainstream smoke between recent results and data
decreased after year 2000. Average NNN levels decreased from
obtained in 2000 are reported in Table 2. The columns of Table 2
987 ng/g tobacco in year 2000 to 266 ng/g tobacco in 2001. From
report observed differences (expressed also in relative terms in-
2002 to 2013, NNN values fluctuated on average between a mini-
side the parentheses), uncertainty on the estimated differences,
mum of 122 ng/g tobacco and a maximum of 471 ng/g tobacco.
simultaneous 95% CIs, and adjusted p-values. P-value adjustment
Similarly, average NNK levels were reduced from 1545 ng/g tobacco
and simultaneous CIs are necessary to correct for multiple testing,
in flue-cured Virginia tobacco to 278 ng/g tobacco in 2001. In
as described in the previous section.
116 I. Gunduz et al. / Regulatory Toxicology and Pharmacology 76 (2016) 113e120

Table 2
Summary results for the statistical assessment of the comparisons for NNN and NNK in tobacco filler and mainstream smoke between results from recent PMI brand surveys
and data from PMI 2000 benchmark study. The observed differences (relative differences are given in parenthesis) together with their standard error (SE) are reported. The last
two columns provide the simultaneous 95% confidence intervals for the observed differences together with the adjusted p-values for testing the hypothesis of no true dif-
ference. Significance levels are given as follows:*p < 0.05,**p < 0.01, and***p < 0.001 (p-values are adjusted using Dunnett test correction).

Constituent [unit] Comparison Observed difference SE difference Simult. 95% CI P-Value (adj)

NNN [ng/g] 2008e2000 765.0 ( 34%) 108 (-1026; 503) 0.000***

2010e2000 884.0 ( 40%) 103 (-1132; 636) 0.000***
2012e2000 638.0 ( 29%) 111 (-906; 369) 0.000***
2014e2000 1251.0 ( 56%) 147 (-1607; 896) 0.000***
NNK [ng/g] 2008e2000 494.6 ( 61%) 29.6 (-565.8; 423.5) 0.000***
2010e2000 473.0 ( 59%) 28.2 (-540.9; 405.2) 0.000***
2012e2000 488.4 ( 60%) 30.3 (-561.2; 415.6) 0.000***
2014e2000 473.9 ( 59%) 39.3 (-568.3; 379.5) 0.000***
NNN [ng/mg SN] 2008e2000 22.6 ( 19%) 6.3 (-37.76; 7.39) 0.001***
2010e2000 38.1 ( 32%) 6 (-52.49; 23.69) 0.000***
2012e2000 21.6 ( 18%) 6.5 (-37.13; 5.97) 0.003***
2014e2000 42.7 ( 36%) 8.5 (-63.28; 22.08) 0.000***
NNK [ng/mg SN] 2008e2000 15.8 ( 22%) 4.3 (-26.14; 5.54) 0.001***
2010e2000 16.0 ( 22%) 4.1 (-25.80; 6.27) 0.000***
2012e2000 12.3 ( 17%) 4.4 (-22.83; 1.70) 0.017**
2014e2000 20.1 ( 28%) 5.8 (-34.10; 6.17) 0.002***
NNN [ng/mg tar] 2008e2000 1.5 ( 20%) 0.4 (-2.436; 0.648) 0.000***
2010e2000 2.5 ( 33%) 0.4 (-3.366; 1.670) 0.000***
2012e2000 2.0 ( 26%) 0.4 (-2.885; 1.051) 0.000***
2014e2000 2.8 ( 37%) 0.5 (-4.035; 1.610) 0.000***
NNK [ng/mg tar] 2008e2000 1.1 ( 24%) 0.2 (-1.667; 0.544) 0.000***
2010e2000 1.1 ( 24%) 0.2 (-1.635; 0.570) 0.000***
2012e2000 1.1 ( 24%) 0.2 (-1.721; 0.570) 0.000***
2014e2000 1.3 ( 29%) 0.3 (-2.103; 0.580) 0.000***

SN e Smoke nicotine.

consecutive years NNK values fluctuated on average from a mini-
mum of 115 ng/g tobacco to a maximum of 506 ng/g tobacco
(Table 3). The linear trend for NNN and NNK in the flue-cured
Virginia tobacco lots was not statistically significant on the 5% er-
ror rate; it was marginally significant at the 10% error rate (the t-
test on the linear regression slope being zero results in a p-value of
0.096 for NNK and 0.103 for NNN).
Data from individual Burley tobacco lots purchased in USA
indicate that average levels of NNN and NNK were reduced sub-
stantially over a 15-year period. Average NNN levels in individual
Burley tobacco lots decreased from 9754 ng/g in 2000e6862 ng/g
in 2001 and fluctuated between a minimum 2990 ng/g to a
maximum 7390 ng/g until 2006. After 2006 average NNN values
fluctuated between a minimum 856 ng/g to a maximum 4022 ng/g.
Average NNK values followed a similar pattern of reduction
(Table 4). The linear trend for both NNN and NNK in the Burley
tobacco lots is statistically significant; the corresponding p-values
are equal to 0.01 for NNN and 0.05 for NNK. Linear trends for both
NNN and NNK are tested on the logarithmic scale in order to meet
linearity.
4. Discussion
Data derived from PMI marketed products over 15 years indicate
that there is, on average, a consistent and substantial reduction of
both NNN and NNK in tobacco filler and mainstream cigarette
smoke of sampled cigarettes. These results are in line with a
recently published decreasing trend of TSNA in mainstream smoke
of US commercial cigarettes analyzed over 35 years (Appleton et al.,
2013). Smoke TSNA data presented in the latter study were
generated by machine smoking cigarettes under the FTC/ISO
smoking conditions and data presented in this study were gener-
ated using HCI machine smoking regimen. Although different ma-
chine smoking regimens were used, relative reduction of NNN and
NNK reported in both studies were consistent.
The reduction of both NNN and NNK in tobacco filler of sampled
cigarettes could be attributed to: 1) Flue-curing barn conversion
which is effective for Virginia tobaccos. 2) Adoption of good agri-
cultural practices including use of fertilization regimes to minimize
TSNA formation and 3) the utilization of LC Burley tobacco seeds
resulting from a seed screening for low conversion of nicotine to
nornicotine, a precursor of NNN. The analysis of flue-cured Virginia
tobacco lots purchased by PMI between 2000 and 2014 indicates
that average NNN and NNK levels decreased noticeably after 2000
when the retrofitting of commercial curing from direct-fired curing
to heat exchanger systems started. After 2000 the average levels of
both NNN and NNK remained at low levels. Similar reductions were
also observed in NNN and NNK in the tobacco filler predominantly
containing flue-cured tobacco grown in Canada and mainstream
smoke of sampled cigarettes after the adoption of heat exchanger in
flue-curing barns (Rickert et al., 2008). Tobacco lots that had NNN
or NNK levels above the limit of detection but not quantifiable were
excluded from our analysis. Therefore, average values of NNN and
NNK provided in this report are overestimated for flue-cured Vir-
ginia tobaccos.
In Burley tobaccos, adoption of good agricultural practices
contributed to an average reduction of both NNN and NNK
(University of Kentucky College of Agriculture, University of
Tennessee Institute of Agriculture, North Carolina State University
and Virginia Tech, 2015). Among the practices mentioned above,
the use of low converter seeds contributed to an average reduction
of NNN due to the lower conversion of nicotine to nornicotine, a
precursor of NNN (Miller et al., 2004; Jack et al., 2007). Adoption of
recommended nitrogen usage and curing practices, including good
ventilation in the Burley curing barns to reduce humidity, should
contribute to the reduction of NNN and NNK. Although data derived
from individual Burley tobacco lots purchased in USA are highly
variable, there is an apparent downward trend both in NNN and
NNK since 2000 (Table 4 and Fig. 1). High variability of TSNA
observed across the years could be as the result of changes in hu-
midity and temperatures during the curing. Under dryer conditions
during curing, TSNA tend to be both lower and less variable, while
 I. Gunduz et al. / Regulatory Toxicology and Pharmacology 76 (2016) 113e120 117

Fig. 1. 95% simultaneous CIs for the difference between NNN and NNK average levels in tobacco filler and mainstream smoke between recent PMI brand surveys and the PMI 2000
benchmark data. The estimated differences and their associated lower and upper limits are expressed in relative terms by dividing them to the corresponding average values of NNN
and NNK reported in the PMI 2000 benchmark study. SN- Smoke nicotine.

Table 3
Average NNN and NNK levels in flue-cured Virginia tobacco purchased by PMI in the USA during the period 2000 to 2014.

Crop year Number of lots analyzed NNN [ng/g] NNK [ng/g]

Number of lots ADL AVG SE Number of lots ADL AVG SE

2000 5 5 987.2 234.2 5 1545.2 349.5

2001 19 19 266.1 53.1 19 278.1 53.8
2002 8 8 324.3 92.5 8 313.1 84.3
2003 5 5 121.5 33.0 5 114.8 27.3
2004 9 9 125.3 12.3 9 115.3 13.9
2005 17 17 262.2 40.1 17 168.7 29.6
2006 51 51 238.5 30.0 51 245.1 54.9
2007 27 27 354.0 50.0 27 287.4 28.9
2008 34 33 470.9 65.9 34 343.3 61.8
2009 30 30 289.2 34.4 30 257.9 43.0
2010 227 199 314.8 12.9 188 494.6 35.5
2011 202 188 400.1 20.8 188 416.1 31.8
2012 152 120 289.5 22.8 113 318.1 28.3
2013 125 12 102.1 11.1 15 119.4 10.1
2014 119 59 226.0 10.7 55 210.9 11.8

SE- Standard error; ADL e Above detection limit.

118 I. Gunduz et al. / Regulatory Toxicology and Pharmacology 76 (2016) 113e120

Table 4
Average NNN and NNK levels in Burley tobacco lots purchased by PMI in the USA during the period 2000 to 2014.

Crop year Number of lots analyzed NNN [ng/g] NNK [ng/g]

Number of lots ADL AVG SE Number of lots ADL AVG SE

2000 11 11 9754.4 261.2 11 1172.9 152.7

2001 13 13 6862.5 642.5 13 1060.9 158.2
2002 5 5 4683.5 316.2 5 517.1 19.5
2003 3 3 3486.5 439.9 3 593.0 26.3
2004 3 3 2989.7 376.5 3 375.7 81.0
2005 12 12 4768.3 553.2 12 717.6 102.9
2006 13 13 7389.9 1760.8 13 1371.7 405.5
2007 16 16 2093.4 75.2 16 390.4 41.5
2008 22 22 1840.6 123.7 22 202.4 16.8
2009 12 12 4021.8 316.5 12 688.5 84.1
2010 123 123 1006.8 49.7 123 414.6 32.7
2011 83 83 856.9 31.3 83 156.3 7.3
2012 101 101 1746.0 72.0 101 306.6 13.9
2013 47 47 2730.4 104.9 47 379.1 13.4
2014 64 64 2334.7 182.7 64 336.1 21.1

SE- Standard error; ADL e Above detection limit.

Fig. 2. Average values and associated 95% confidence intervals for NNN (in filled circles) and NNK (in filled triangles) in US Burley tobacco lots over the time period 2000 to 2014.
The left Y-axis is used for NNN; the right Y-axis is used for NNK.

higher humidity during curing leads to greater variability and over
all higher TSNA in tobacco (Fisher et al., 2012). The high variability
of TSNA in Burley tobacco was also observed in a survey conducted
between 1990 and 1998 indicating that TSNA values in Burley to-
bacco samples from USA vary between ~4000 ng/g to 34000 ng/g
(d'Andres et al., 2003). The smaller variability of NNN and NNK
levels observed across years after 2006 could possibly be due to the
better adoption of agricultural practices and use of LC Burley to-
bacco seeds.
Our data suggest that, since 2008, a significant reduction was
observed in NNN and NNK yields in MS cigarette smoke compared
to the PMI 2000 benchmark study data, however, the reduction was
not as pronounced as the reduction observed in tobacco filler of
sampled cigarettes (Table 2). While in average 59e61% reduction of
NNK is observed in tobacco filler, only 17e29% reduction of NNK is
observed in mainstream cigarette smoke per mg of nicotine of
cigarettes sampled in 2008 and later compared to the PMI 2000
benchmark study data. A similar trend but smaller difference was
also observed for NNN. The amounts of NNN and NNK in main-
stream smoke are a combination of direct transfer from tobacco and
of pyro-synthesis and thermal release during cigarette combustion.
It was recently demonstrated that matrix-bound NNK which ac-
counts for a large portion of total NNK in Burley and flue-cured
Virginia tobacco could be released at above 200  C and represent
a large proportion of NNK in mainstream cigarette smoke (Lang and
Vuarnoz, 2015). Analytical methods used in our study cannot detect
matrix-bound NNK, therefore NNK measured in tobacco is under-
estimated and does not represent total NNK. It was reported
(Djordjevic et al., 1991) that spiking cigarettes with nornicotine
increased NNN in MS smoke by 27% potentially indicating a pyro-
synthesis of NNN resulting from most likely the nitrosation of
nornicotine during combustion. Variation of smoke and tobacco
filler NNN of sampled cigarettes appeared to be correlated among
different years (Table 2) confirming that tobacco filler NNN is the
major contributor of variation of smoke NNN, as already shown
(d'Andres et al., 2003; Hyodo et al., 2015).
The selective reduction of smoke constituents in conventional
cigarettes is very challenging since a decrease of one smoke con-
stituent may be accompanied by an increase of other smoke con-
stituents (King et al., 2007; Piade et al., 2013). Precisely, while
 I. Gunduz et al. / Regulatory Toxicology and Pharmacology 76 (2016) 113e120
Fig. 3. Average values and associated 95% confidence intervals for NNN (in filled circles) and NNK (in filled triangles) in US flue-cured Virginia tobacco lots over the time period
2000 to 2014. The left Y-axis is used for NNN; the right Y-axis is used for NNK.
reductions were identified for NNN and NNK levels, an increase in
the formaldehyde yields in MS smoke was observed during the
same time period (data not shown). The adoption of recommended
nitrogen usage for the fertilization that aims to reduce TSNA, has
the potential to decrease the ammonia and nitrogen oxide in
mainstream smoke. Lower ammonia and nitrogen oxide in the MS
smoke could therefore cause an increase of formaldehyde (Paine
et al., 2004; Baker et al., 2006; Glarborg et al., 2003).
The results obtained since 2000 shall be considered in light of
several limitations.
1 Study design: The ideal study to address the question of trends
in TSNA levels in mainstream smoke of cigarettes over time
would be a longitudinal study spanning many years, employing
a single cigarette design manufactured over many tobacco crop
years, and using the same validated and standardized methods
to analyze all samples over the course of the study (Appleton
et al., 2013). The data considered in this study came from two
different sources, namely the PMI 2000 benchmark and PMI
market survey studies and have sufficient consistency to derive
relevant information regarding to the evolution of the TSNA
levels in marketed products during the past 15 years. The ele-
ments of consistency are as follows a) the sampling methodol-
ogy used for the PMI 2000 benchmark study and PMI studies
were similar, namely they aimed covering wide range of design
characteristics and of tar yields of PMI products that are avail-
able within different regions. b) The same analytical laboratory
and methods were used for all studies to analyze mainstream
smoke and tobacco filler of commercial cigarettes. Considering
those elements of consistency and the number of samples
available for each year, general trends about the evolution of
TSNA could be derived over the period of 2000e2014, by
comparing the average levels from the different samples ob-
tained each year.
2 Analytical variability of methods used for TSNA analysis:
Although the analyses of cigarette samples for the PMI 2000
benchmark study and the PMI internal surveys were performed
within the same laboratory to avoid potential systematic bias
due to methods from different laboratories, the variability over
time within one laboratory for the same product can be rela-
tively high (Oldham et al., 2014; Belushkin et al., 2015): For NNN,
119
a difference of 61% was calculated between the lowest and
highest smoke NNN yields for a monitor cigarette (Purkis et al.,
2012). Standard errors associated with our individual data sets
are provided in Table 1. In comparison to 2000 data, and despite
the high variability of the data, consistent decrease of NNN and
NNK in tobacco fillers and mainstream smoke of cigarettes
sampled from 2008 to 2014 indicates that this decrease is not
only due to temporal variability.
3 Sample size of individual tobacco lots analyzed over the years:
Lower number of samples analyzed from tobacco lots purchased
in USA by PMI in some years may not be representative from the
whole crop. It provides nevertheless a good indication and
corroborates with the data derived from tobacco filler of ciga-
rette samples analyzed across the years.
4 Limitation of methods used to detect NNK concentration in to-
bacco: The method used in this study can only measure free
NNK which may only represents 23e47% of NNK in Burley and
Flue-Cured Virginia tobaccos respectively (Lang and Vuarnoz,
2015). Therefore NNK trends observed in smoke are more
representative.
In conclusion, review of data covering 15 years timespan indi-
cate that both NNN and NNK levels decreased over time in Burley
and flue-cured Virginia tobacco lots, as well as in cigarette tobacco
filler and mainstream smoke of sampled cigarettes. The decreasing
trend coincides with the implementation of various initiatives put
in place by industry and agricultural community to reduce TSNA.
Many public health experts and PMI believe that selective reduc-
tion of smoke constituents in conventional cigarettes that burn
tobacco is unlikely to reduce the risk of tobacco related diseases.
Nonetheless, PMI requires that leaf suppliers and tobacco growers
adhere to 1) Use of heat exchanger in flue-curing barns, 2) Good
agricultural practices including use of fertilization regimes to
minimize TSNA formation and 3) The utilization of low converter
Burley tobacco seeds, in order to minimize TSNAs in flue-cured
Virginia and Burley tobaccos.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.yrtph.2016.01.012.
120 I. Gunduz et al. / Regulatory Toxicology and Pharmacology 76 (2016) 113e120

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