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Reductions in the tobacco specific nitrosamine (TSNA) content of tobaccos taken from commercial Canadian cigarettes and corresponding reductions in TSNA deliveries in mainstream smoke from such cigarettes
Regulatory Toxicology and Pharmacology 51 (2008) 306–310
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Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Reductions in the tobacco specific nitrosamine (TSNA) content of tobaccos taken from commercial Canadian cigarettes and corresponding reductions in TSNA deliveries in mainstream smoke from such cigarettes W.S. Rickert a,*, P.J. Joza a, M. Sharifi a, J. Wu a, J.H. Lauterbach b a b
Labstat International, ULC, 262 Manitou Drive, Kitchener, Ont., Canada N2C 1L3 Lauterbach & Associates, LLC, 211 Old Club Court, Macon, GA 31210-4708, USA
a r t i c l e
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Article history: Received 9 November 2007 Available online 24 April 2008 Keywords: TSNA Tobacco Commercial cigarettes Canadian cigarettes Mainstream smoke
a b s t r a c t Tobacco specific nitrosamines (TSNAs) are suspected to cause smoking-related neoplastic diseases. The change from direct-fired to indirect-fired barns (aka kilns) for curing bright (aka Virginia, flue-cured) tobaccos was made to reduce the TSNA concentrations. The effectiveness of such processes in reducing the deliveries of TSNAs to the users of the products should be monitored. However, it is difficult to assess the effects of this reduction on the TSNA levels in mainstream smoke when cigarette blends contain burley tobaccos and other blend components that can increase smoke TSNA concentrations. Canadian cigarettes made prior to and in the few years just after the conversion to indirect-fired curing should not be subject to such interferences. Thus, the TSNA content of tobaccos and mainstream smoke from six brands of Canadian cigarettes produced in 2003, 2004, and 2005 were determined. Reductions in NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], the most important TSNA in flue-cured tobaccos, levels in the tobacco blends ranged from 60% to 85%. The corresponding reductions in mainstream smoke TSNA levels ranged from 59% to 72% (ISO smoking conditions) and 58–76% (Health Canada Intensive smoking conditions). These results show that other factors (microorganisms, nitrite levels) may be negating the TSNA reductions achieved by indirect-fired curing. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Canadian cigarettes have historically been unique, as the blend has been composed predominately of flue-cured tobaccos grown in southern Ontario (Physicians for a Smoke-free Canada, 1999). Thus, such cigarettes have been different from the major brands of fluecured cigarettes on international markets as those likely have contained tobaccos from several of the world’s flue-cured tobacco growing regions. Moreover, it is unlikely that the major Canadian brand styles have contained burley or other air-cured tobaccos. Furthermore, with the exception of the mentholated brands, most manufactured Canadian cigarettes are believed to be additive-free. A listing of additives and processing aids used in many Canadian cigarette brand styles made from 1999 to 2003 (and for some brands, 2004) can be found at web site run by the Canadian province of British Columbia (British Columbia, 2007). Thus, there is likely a more direct connection between tobacco chemistry and smoke chemistry than there would be in many other cigarette markets. However, this may change because of steps taken by the Canadian and Ontario governments to make tobacco farming very * Corresponding author. Fax: +1 519 748 1654. E-mail address: [email protected] (W.S. Rickert). 0273-2300/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2008.04.009
unattractive (Ramsey and Smit, 2001, 2002). If this takes place, then manufacturers may source the tobaccos from other growing regions, and the consistency of the tobacco chemistries may change. Furthermore, Imperial Tobacco Canada, which is the largest of the three Canadian tobacco companies, has closed its Canadian factories and has moved its manufacturing operations to Monterrey, Mexico (Imperial Tobacco Canada, 2005; Kirsch, 2008). Consequently, it is important to document the tobacco and smoke chemistries of Canadian cigarettes before there are significant changes in the products. Tobacco specific nitrosamines (TSNAs) are viewed by many health experts as one of the leading causes of smoking-related neoplastic diseases (Gray and Boyle, 2004). One of the TSNAs, NNK [4(methylnitrosamino)-1-(3-pyridyl)-1-butanone], is believed to be one of the most potent carcinogens in cigarette smoke (Hecht, 1999). NNK and the other TSNAs have not been found in green (on the living plant) tobacco except in special circumstances (Chamberlain et al., 1986; Brunnemann and Hoffmann, 1991). They are formed mainly during curing and processing of the tobacco (Brunnemann and Hoffmann, 1991; Baker, 1999). Additional amounts are also formed in the burning cigarette through pyrosynthesis (Baker, 1999; Moldoveanu et al., 2001, 2004). From a toxicological point of view, the two TSNAs of concern are NNK, as noted
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above and found in highest amounts in flue-cured tobaccos, and NNN (N0 -nitrosonornicotine) found in highest amounts in air-cured tobaccos (Brunnemann and Hoffmann, 1991; Baker, 1999). In their symposium paper at the 2001 Tobacco Science Research Conference, Peele and co-workers reported on their studies that began in 1998 and showed the levels of NNK in flue-cured tobacco could be reduced by reducing the concentration of nitrogen oxides (NOx) in the atmosphere around the leaves in the curing barn (Peele et al., 2001). This method for NNK reduction, which was first made public in 1999, was accomplished by use of heat exchangers to isolate the combustion gases from the heated air around the leaves (Peele and Gentry, 1999). These indirect-fired barns were the modern equivalent of older indirect-fired barns that had been used prior to the rise in energy costs. When energy costs began to increase in the 1960s, farmers in the US had switched to the more efficient direct-fired barns (Peele et al., 2001). Canadian farmers also changed to direct-fired barns (known in Canada as kilns). Once the benefits of indirect-fired barns became known, those concerned with the production of flue-cured tobacco in the United States developed a program to convert the direct-fired barns to indirect-fired ones (Hamm, 2001). Over three hundred samples were collected in 2000 and analyzed for TSNAs. TSNA levels were found to be 93% less overall for tobaccos cured in indirect-fired barns compared with those cured in direct-fired barns; and the reduction was found for all four TSNAs determined, not just NNK (Boyette and Hamm, 2001). Decreases in the TSNA content of flue-cured tobaccos grown in Canada were not observed until 2001 (Marchand et al., 2005). In that year, the Ontario government provided $20 million Canadian dollars to the farmers to convert all their kilns to indirect heating as required by the Canadian tobacco industry (Physicians for a Smoke-free Canada, 2006). Because of the importance given to TSNA reduction by some authorities in smoking-related diseases, and because Canadian cigarettes historically have been fabricated from flue-cured tobaccos grown in Ontario, we decided to see if the use of indirect-fired curing barns resulted in a decrease TSNA mainstream smoke deliveries. We had available Canadian-manufactured cigarettes that had been produced in 2003, 2004, and 2005. In addition, tobacco and mainstream smoke TSNA data from these cigarettes would likely provide a baseline on these toxicants before significant changes in the sourcing and manufacture of tobacco products had occurred. We determined the TSNA levels in the tobaccos taken from six brands of Canadian cigarettes produced in 2003, 2004, and 2005 and the mainstream TSNA deliveries under both ISO and Health Canada Intensive smoking conditions (Wu et al., 2008). The remainder of this report deals with the results of the analytical determinations and the extent of the reductions in TSNAs in the tobacco blends and cigarette mainstream smoke (MSS). 2. Materials and methods 2.1. Cigarettes Six brands of commercially available Canadian flue-cured filter cigarettes produced in 2003, 2004 and 2005 were used for this study. The brands are described using the declared values from the packaging and the total cigarette length (Table 1). On receipt, all cigarettes were stored at 4 °C until laboratory use. Cigarettes were conditioned according to International Organization for Standardization (ISO) conditioning regimen prior to analyses (ISO Standard 3402, 1999a). 2.2. Mainstream smoke collection and determination of TSNAs in mainstream smoke The mainstream smoke collections were done under ISO (ISO Standard 3308, 2000a; ISO Standard 4387, 2000b) and Health Canada Intensive (Health Canada Method T-115, 1999a) conditions. Seven replicates for each test brand from each year of the study were analyzed for TSNA content with a LC-MS/MS (liquid chromatography with tandem mass spectrometry) method that uses an isotopically labeled internal standard for each of the four TSNAs determined (Wu et al., 2008). ISO smoking conditions call for a 35 mL puff of 2 s duration to be taken every minute
Table 1 Cigarette samples used in this study Brand
Nominal size and cigarette length (mm)
ISO Tar (mg/cig)
Health Canada Intensive Tar (mg/cig)
A B C D E F
King (85) King (85) Regular (72) King (85) Regular (72) Regular (72)
4 9 10 12 13 15
25 29 29 31 33 33
until the burning or smoldering tobacco (coal) reaches the burn line. There is no blocking of filter ventilation. Health Canada Intensive smoking conditions call for a 55 mL puff of 2 s duration to be taken every 30 s until the coal reaches the burn line. Filter ventilation is completely blocked. 2.3. Other mainstream smoke analytes Other smoke analytes such as TPM (total particulate matter), water, nicotine, CO (carbon monoxide), benzene, and B[a]P (benzo[a]pyrene) were also determined on the MSS from the cigarettes for comparison with prior results (TPM, water, nicotine, and CO) and to determine if the transition to indirect-fired curing had caused unexpected changes in other important smoke analytes such as benzene and B[a]P. ISO determinations of TPM, water, nicotine, and carbon monoxide were done according to ISO Standards (ISO Standard 4387, 2000b; ISO Standard 10362-1, 1999b; ISO Standard 10315, 2000c; ISO Standard 8454, 1995). Mainstream smoke benzene and B[a]P deliveries were determined using Health Canada methods (Health Canada T-116, 1999b; Health Canada T-103, 1999c). 2.4. Determination of TSNAs in cigarette tobaccos The determination of TSNA content of cigarette tobaccos was conducted using three replicate samples from each test brand of each year of the study. The tobacco part of the LC-MS/MS method was used (Wu et al., 2008).
3. Results and discussion 3.1. Results from TSNA determinations The results from the determinations of TSNAs in the cigarette tobaccos from the six brands are shown in Table 2. Results from the determinations of the TSNAs in mainstream cigarette smoke under ISO and Health Canada Intensive conditions are shown in Tables 3 and 4, respectively. The data showed that decreases in tobacco TSNA levels from 2003 to 2005 ranged from around 80% for Brand A to around 60%
Table 2 Results of determinations of TSNAsa in cigarette tobacco Sample ID
Year
NNN (ng/ g)
NAT (ng/ g)
NAB (ng/ g)
NNK (ng/ g)
Total TSNA (ng/g)
A A A B B B C C C D D D E E E F F F
2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005
487 365 105 530 375 152 441 425 143 482 479 159 464 410 164 532 386 139
570 432 131 589 490 204 548 456 169 602 603 192 600 494 221 654 473 193
27.5 16.4 6.25 29.1 19.8 7.45 24.3 19.9 8.42 28.2 24.0 9.63 27.6 21.5 11.2 29.1 19.7 8.36
714 478 110 777 601 222 648 618 185 759 718 227 700 678 281 800 615 219
1799 1291 352 1925 1486 585 1661 1519 505 1871 1824 588 1792 1604 677 2015 1494 559
a NNN, N0 -nitrosonornicotine; NAT, N0 -nitrosoanatabine; NAB, N0 -nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
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Table 3 Results of determinations of TSNAsa in MSSb under ISO conditions
Table 5 Percentage reductions for TSNAsa in tobacco and smoke from 2003 to 2005
Sample ID
Year
NNN (ng/cig)
NAT (ng/cig)
NAB (ng/cig)
NNK (ng/cig)
Brand
Sampleb
NNN (%)
NAT (%)
NAB (%)
NNK (%)
A A A B B B C C C D D D E E E F F F
2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005
9.43 7.05 3.31 18.5 13.4 6.83 18.4 14.4 6.51 24.3 20.1 7.43 20.8 16.2 8.89 22.7 17.1 9.14
13.3 9.92 5.43 26.4 20.7 10.0 24.3 21.4 10.9 34.3 30.5 14.3 28.1 23.1 13.8 34.8 28.4 14.5
1.01 0.700 0.398 1.61 1.06 0.525 1.72 0.98 0.482 2.32 1.62 0.655 1.93 1.11 0.660 2.48 1.21 0.714
15.6 11.7 4.32 33.6 25.7 10.2 30.7 22.4 10.0 37.9 36.4 13.1 35.2 28.2 14.6 41.2 32.0 14.5
A A A B B B C C C D D D E E E F F F
TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT
78.4 64.9 70.9 71.4 63.1 66.4 67.6 64.6 64.2 67.0 69.4 68.5 64.8 57.2 63.9 73.9 59.7 71.8
77.1 59.1 66.2 65.4 62.2 63.3 69.1 54.9 61.5 68.1 58.3 62.6 63.1 50.9 57.3 70.5 58.2 67.3
77.3 60.4 53.3 74.4 67.4 57.0 65.4 72.0 51.9 65.8 71.7 70.2 59.6 65.7 51.5 71.2 71.2 67.8
84.6 72.2 76.3 71.4 69.5 69.6 71.4 67.4 68.4 70.1 65.4 67.8 59.9 58.5 58.3 72.7 64.9 68.0
a NNN, nitrosonornicotine; NAT, nitrosoanatabine; NAB, nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. b MSS, mainstream cigarette smoke.
Table 4 Results of determinations of TSNAsa in MSSb under HC INTc conditions Sample ID
Year
NNN (ng/cig)
NAT (ng/cig)
NAB (ng/cig)
NNK (ng/cig)
A A A B B B C C C D D D E E E F F F
2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005
34.6 27.5 10.1 47.6 33.9 16.0 36.1 30.8 12.9 52.1 40.8 16.4 38.6 31.6 13.9 47.2 32.9 13.3
54.2 41.9 18.3 70.2 51.5 25.8 57.0 42.3 22.0 75.7 62.7 28.3 58.7 46.9 25.1 73.4 50.6 24.0
2.71 1.81 1.26 3.57 2.21 1.54 2.76 2.26 1.33 4.26 2.86 1.27 3.02 2.23 1.47 4.03 2.31 1.30
60.6 45.3 14.4 78.4 62.9 23.8 63.3 51.4 20.0 84.5 76.8 27.2 69.5 59.7 29.0 81.8 61.2 26.2
a
NNN, nitrosonornicotine; NAT, nitrosoanatabine; NAB, nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. b MSS, mainstream cigarette smoke. c HC INT, Health Canada Intensive smoking conditions.
for Brand E (Table 5). The data presented by Marchand and coworkers indicated that TSNA levels in Canadian cigarette products decreased about 40% from 2003 to 2004 (Marchand et al., 2005). Another conclusion that can be drawn from the data in Table 5 is that the reductions in MSS TSNA deliveries were reflective of the reductions observed for tobacco TSNAs. However, the percentage reductions observed for MSS TSNA deliveries were generally less than those observed for the tobacco TSNAs. This phenomenon was also noted by Marchand et al. (2005). In addition, they reported that transfer rates for the TSNA were less than 10% so even a large change in tobacco concentration is not reflected fully in mainstream deliveries. There is no reason to believe that the percentage reductions in MSS TSNA deliveries between 2003 and 2005 product should be any different for ISO smoking conditions than they are for Health Canada Intensive smoking conditions. No significant changes were found in the other MSS analytes that were determined. This was not unexpected since the difference in curing was not expected to change the tobacco significantly other than to reduce the TSNA levels. Thus, the results of these
a NNN, nitrosonornicotine; NAT, nitrosoanatabine; NAB, nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. b TOB, tobacco taken from cigarettes; ISO, ISO smoking conditions; HC INT, Health Canada Intensive smoking conditions.
determinations are not included in this article. Very recently, Hayes and co-workers reported that experimental cigarettes made with indirect-fired flue-cured tobaccos gave significantly lower mainstream smoke acetone deliveries (353.9 ± 5.2 lg/cig versus 368.9 ± 7.3 lg/cig) and significantly higher smoke nicotine deliveries (2.35 ± 0.08 mg/cig versus 2.21 ± 0.01 mg/cig) than did cigarettes prepared with direct-fired flue-cured tobaccos (Hayes et al., 2007). Unfortunately, those authors did not provide analytical data on the cigarettes and tobaccos taken from those cigarettes. On the basis of the available information, we do not believe the differences in MSS nicotine and MSS acetone reported by Hayes and co-workers indicated changes in product chemistry caused by indirect-fired barns other than the reduction in TSNA concentrations. 3.2. Discussion of results In 1990, Fischer and co-workers reported on the tobacco TSNA contents and MSS smoke yields (ISO) for twenty-five brands styles of Canadian cigarettes (Fischer et al., 1990). They reported tobacco TSNA levels as follows: NNN, 256–979 ng/cig; NNK, 465–878 ng/ cig; and NAB+NAT, 572–1033 ng/cig. Assuming the cigarettes Fischer and co-workers analyzed contained 750 mg of tobacco, the TSNA levels they reported would be similar to those reported by Marchand and co-workers for cigarettes produced in 2000 (Marchand et al., 2005). More recently, Morin and co-workers reported that when they cured Canadian tobacco (1999 crop) under direct-fired conditions, total TSNA levels approached 2 ppm (2000 ng/g), but when tobaccos from the same lots were cured using indirect heating, total TSNA levels were on the order of 0.25–0.35 ppm (Morin et al., 2004). Our results indicated that total TSNA levels ranged from 1.7 to 2.0 ppm for 2003 products and 0.35 to 0.68 ppm for 2005 products. Those levels are on the same order of magnitude that Joza and co-workers reported for the tobaccos taken from cigarettes obtained in 1970 (average of 1.12 ppm) and maintained in secure storage (Joza et al., 2000). Thus, the return to indirect-firing has reduced TSNA levels to about the same levels found in product made in 1970. Based on the report by Boyette and Hamm on US-grown fluecured tobaccos (2001), lower levels would have been expected even though the US had been known for having high TSNA levels in its flue-cured tobaccos (d’Andres et al., 2003). This raises the question of other factors that might need to be considered in pro-
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ducing Canadian-style cigarettes with even lower TSNA levels. Other factors suggested have included microbial populations on the leaf (Morin et al., 2004), barn rot (Spurr and Hamm, 2003), and nitrite levels in the tobacco (Wanfeng and Yuanying, 2002). More recently, there has been a presentation on the effects of TSNA formation during the manufacture of reconstituted tobaccos (Rigoulay et al., 2006) and another presentation showing how temperature and added nitrite and nitrate increase TSNA levels in fluecured tobaccos (Fannin et al., 2006). Very recently, mainstream smoke nitric oxide concentrations have been correlated with TSNA deliveries (Hyodo et al., 2007). Taken altogether, the information in from those sources would indicate that the use of stem and/or reconstituted tobaccos in flue-cured blends could increase the TSNA concentrations above those found in the tobaccos sampled right after curing in indirect-fired barns. Continual efforts should be made to reduce TSNAs concentrations to toxicologically insignificant levels, if possible. Flue-cured tobaccos have been found with less than 30 ng/g of each TSNA and less than 100 ng/g total TSNA (Renaud and Zuber, 1996). The factors in addition to use of indirect heating for the curing need to be identified and reduced to practice, so that the concentrations of TSNAs in leaf, blend, and smoke can be minimized.
4. Conclusions We have documented the TSNA levels in the mainstream smoke from commercial cigarettes picked-up in the Canadian marketplace in 2003, 2004, and 2005. We have also documented the TSNA levels in the tobaccos taken from the same populations of cigarettes used for the smoke determinations. The decreases in MSS TSNA smoke deliveries followed the decreases in tobacco TSNA concentrations, which, in turn, were caused by the change from direct-fired to indirect-fired curing of the tobacco. However, the percentage decreases in MSS TSNA deliveries were not as great as the percentage decreases for the corresponding tobaccos. This finding shows the need for further work on understanding the relationships among tobacco TSNA concentrations after curing, tobacco processing during cigarette manufacture, and MSS TSNA deliveries. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yrtph.2008.04.009. References Baker, R.R., 1999. Smoke chemistry. In: Davis, D.L., Nielsen, M.T. (Eds.), Tobacco Production, Chemistry, and Technology. Blackwell Science Inc, Malden, MA, pp. 425–427. Boyette, M.D., Hamm, L.A., 2001. Results of the Year 2000 TSNA sampling program in flue-cured tobacco. Rec. Adv. Tob. Sci. 27, 17–22. British Columbia, 2007. What is in Cigarettes? Ministry of Health, Government of British Columbia. http://www.healthservices.gov.bc.ca/cgi-bin/ttdr_brand_ search.cgi?constituent = all (accessed February 23, 2008). Brunnemann, K.D., Hoffmann, D., 1991. Analytical studies on N-nitrosamines in tobacco and tobacco smoke. Rec. Adv. Tob. Sci. 17, 71–112. Chamberlain, W.J., Baker, J.L., Chortyk, O.T., Stephenson, M.G., 1986. Studies on the reduction of nitrosamines in tobacco. Tob. Sci. 30, 81–82. d’Andres, S., Boudoux, R., Renaud, J.-M., Zuber, J., 2003. TSNA levels in the mainstream smoke of simplified blend prototypes. Beitr. Tabakforsch. Int. 20, 331–340. Fannin, F.F., Li, X., Burton, H.R., D’Angelo, E., Bush, L., 2006. TSNA changes with cured powdered tobaccos. 60th Tobacco Science Research Conference, Montreal, Canada, September 17–20, Paper #39. Fischer, S., Castonguay, A., Kaiserman, M., Spiegelhalder, B., Preussmann, R., 1990. Tobacco-specific nitrosamines in Canadian cigarettes. Cancer Res. Lin. Oncol. 116, 563–568. Gray, N., Boyle, P., 2004. The case of the disappearing nitrosamines: a potentially global phenomenon. Tob. Control 13, 13–16.
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Reductions in the tobacco specific nitrosamine (TSNA) content of tobaccos taken from commercial Canadian cigarettes and corresponding reductions in TSNA deliveries in mainstream smoke from such cigarettes W.S. Rickert a,*, P.J. Joza a, M. Sharifi a, J. Wu a, J.H. Lauterbach b a b
Labstat International, ULC, 262 Manitou Drive, Kitchener, Ont., Canada N2C 1L3 Lauterbach & Associates, LLC, 211 Old Club Court, Macon, GA 31210-4708, USA
a r t i c l e
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Article history: Received 9 November 2007 Available online 24 April 2008 Keywords: TSNA Tobacco Commercial cigarettes Canadian cigarettes Mainstream smoke
a b s t r a c t Tobacco specific nitrosamines (TSNAs) are suspected to cause smoking-related neoplastic diseases. The change from direct-fired to indirect-fired barns (aka kilns) for curing bright (aka Virginia, flue-cured) tobaccos was made to reduce the TSNA concentrations. The effectiveness of such processes in reducing the deliveries of TSNAs to the users of the products should be monitored. However, it is difficult to assess the effects of this reduction on the TSNA levels in mainstream smoke when cigarette blends contain burley tobaccos and other blend components that can increase smoke TSNA concentrations. Canadian cigarettes made prior to and in the few years just after the conversion to indirect-fired curing should not be subject to such interferences. Thus, the TSNA content of tobaccos and mainstream smoke from six brands of Canadian cigarettes produced in 2003, 2004, and 2005 were determined. Reductions in NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], the most important TSNA in flue-cured tobaccos, levels in the tobacco blends ranged from 60% to 85%. The corresponding reductions in mainstream smoke TSNA levels ranged from 59% to 72% (ISO smoking conditions) and 58–76% (Health Canada Intensive smoking conditions). These results show that other factors (microorganisms, nitrite levels) may be negating the TSNA reductions achieved by indirect-fired curing. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Canadian cigarettes have historically been unique, as the blend has been composed predominately of flue-cured tobaccos grown in southern Ontario (Physicians for a Smoke-free Canada, 1999). Thus, such cigarettes have been different from the major brands of fluecured cigarettes on international markets as those likely have contained tobaccos from several of the world’s flue-cured tobacco growing regions. Moreover, it is unlikely that the major Canadian brand styles have contained burley or other air-cured tobaccos. Furthermore, with the exception of the mentholated brands, most manufactured Canadian cigarettes are believed to be additive-free. A listing of additives and processing aids used in many Canadian cigarette brand styles made from 1999 to 2003 (and for some brands, 2004) can be found at web site run by the Canadian province of British Columbia (British Columbia, 2007). Thus, there is likely a more direct connection between tobacco chemistry and smoke chemistry than there would be in many other cigarette markets. However, this may change because of steps taken by the Canadian and Ontario governments to make tobacco farming very * Corresponding author. Fax: +1 519 748 1654. E-mail address: [email protected] (W.S. Rickert). 0273-2300/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2008.04.009
unattractive (Ramsey and Smit, 2001, 2002). If this takes place, then manufacturers may source the tobaccos from other growing regions, and the consistency of the tobacco chemistries may change. Furthermore, Imperial Tobacco Canada, which is the largest of the three Canadian tobacco companies, has closed its Canadian factories and has moved its manufacturing operations to Monterrey, Mexico (Imperial Tobacco Canada, 2005; Kirsch, 2008). Consequently, it is important to document the tobacco and smoke chemistries of Canadian cigarettes before there are significant changes in the products. Tobacco specific nitrosamines (TSNAs) are viewed by many health experts as one of the leading causes of smoking-related neoplastic diseases (Gray and Boyle, 2004). One of the TSNAs, NNK [4(methylnitrosamino)-1-(3-pyridyl)-1-butanone], is believed to be one of the most potent carcinogens in cigarette smoke (Hecht, 1999). NNK and the other TSNAs have not been found in green (on the living plant) tobacco except in special circumstances (Chamberlain et al., 1986; Brunnemann and Hoffmann, 1991). They are formed mainly during curing and processing of the tobacco (Brunnemann and Hoffmann, 1991; Baker, 1999). Additional amounts are also formed in the burning cigarette through pyrosynthesis (Baker, 1999; Moldoveanu et al., 2001, 2004). From a toxicological point of view, the two TSNAs of concern are NNK, as noted
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above and found in highest amounts in flue-cured tobaccos, and NNN (N0 -nitrosonornicotine) found in highest amounts in air-cured tobaccos (Brunnemann and Hoffmann, 1991; Baker, 1999). In their symposium paper at the 2001 Tobacco Science Research Conference, Peele and co-workers reported on their studies that began in 1998 and showed the levels of NNK in flue-cured tobacco could be reduced by reducing the concentration of nitrogen oxides (NOx) in the atmosphere around the leaves in the curing barn (Peele et al., 2001). This method for NNK reduction, which was first made public in 1999, was accomplished by use of heat exchangers to isolate the combustion gases from the heated air around the leaves (Peele and Gentry, 1999). These indirect-fired barns were the modern equivalent of older indirect-fired barns that had been used prior to the rise in energy costs. When energy costs began to increase in the 1960s, farmers in the US had switched to the more efficient direct-fired barns (Peele et al., 2001). Canadian farmers also changed to direct-fired barns (known in Canada as kilns). Once the benefits of indirect-fired barns became known, those concerned with the production of flue-cured tobacco in the United States developed a program to convert the direct-fired barns to indirect-fired ones (Hamm, 2001). Over three hundred samples were collected in 2000 and analyzed for TSNAs. TSNA levels were found to be 93% less overall for tobaccos cured in indirect-fired barns compared with those cured in direct-fired barns; and the reduction was found for all four TSNAs determined, not just NNK (Boyette and Hamm, 2001). Decreases in the TSNA content of flue-cured tobaccos grown in Canada were not observed until 2001 (Marchand et al., 2005). In that year, the Ontario government provided $20 million Canadian dollars to the farmers to convert all their kilns to indirect heating as required by the Canadian tobacco industry (Physicians for a Smoke-free Canada, 2006). Because of the importance given to TSNA reduction by some authorities in smoking-related diseases, and because Canadian cigarettes historically have been fabricated from flue-cured tobaccos grown in Ontario, we decided to see if the use of indirect-fired curing barns resulted in a decrease TSNA mainstream smoke deliveries. We had available Canadian-manufactured cigarettes that had been produced in 2003, 2004, and 2005. In addition, tobacco and mainstream smoke TSNA data from these cigarettes would likely provide a baseline on these toxicants before significant changes in the sourcing and manufacture of tobacco products had occurred. We determined the TSNA levels in the tobaccos taken from six brands of Canadian cigarettes produced in 2003, 2004, and 2005 and the mainstream TSNA deliveries under both ISO and Health Canada Intensive smoking conditions (Wu et al., 2008). The remainder of this report deals with the results of the analytical determinations and the extent of the reductions in TSNAs in the tobacco blends and cigarette mainstream smoke (MSS). 2. Materials and methods 2.1. Cigarettes Six brands of commercially available Canadian flue-cured filter cigarettes produced in 2003, 2004 and 2005 were used for this study. The brands are described using the declared values from the packaging and the total cigarette length (Table 1). On receipt, all cigarettes were stored at 4 °C until laboratory use. Cigarettes were conditioned according to International Organization for Standardization (ISO) conditioning regimen prior to analyses (ISO Standard 3402, 1999a). 2.2. Mainstream smoke collection and determination of TSNAs in mainstream smoke The mainstream smoke collections were done under ISO (ISO Standard 3308, 2000a; ISO Standard 4387, 2000b) and Health Canada Intensive (Health Canada Method T-115, 1999a) conditions. Seven replicates for each test brand from each year of the study were analyzed for TSNA content with a LC-MS/MS (liquid chromatography with tandem mass spectrometry) method that uses an isotopically labeled internal standard for each of the four TSNAs determined (Wu et al., 2008). ISO smoking conditions call for a 35 mL puff of 2 s duration to be taken every minute
Table 1 Cigarette samples used in this study Brand
Nominal size and cigarette length (mm)
ISO Tar (mg/cig)
Health Canada Intensive Tar (mg/cig)
A B C D E F
King (85) King (85) Regular (72) King (85) Regular (72) Regular (72)
4 9 10 12 13 15
25 29 29 31 33 33
until the burning or smoldering tobacco (coal) reaches the burn line. There is no blocking of filter ventilation. Health Canada Intensive smoking conditions call for a 55 mL puff of 2 s duration to be taken every 30 s until the coal reaches the burn line. Filter ventilation is completely blocked. 2.3. Other mainstream smoke analytes Other smoke analytes such as TPM (total particulate matter), water, nicotine, CO (carbon monoxide), benzene, and B[a]P (benzo[a]pyrene) were also determined on the MSS from the cigarettes for comparison with prior results (TPM, water, nicotine, and CO) and to determine if the transition to indirect-fired curing had caused unexpected changes in other important smoke analytes such as benzene and B[a]P. ISO determinations of TPM, water, nicotine, and carbon monoxide were done according to ISO Standards (ISO Standard 4387, 2000b; ISO Standard 10362-1, 1999b; ISO Standard 10315, 2000c; ISO Standard 8454, 1995). Mainstream smoke benzene and B[a]P deliveries were determined using Health Canada methods (Health Canada T-116, 1999b; Health Canada T-103, 1999c). 2.4. Determination of TSNAs in cigarette tobaccos The determination of TSNA content of cigarette tobaccos was conducted using three replicate samples from each test brand of each year of the study. The tobacco part of the LC-MS/MS method was used (Wu et al., 2008).
3. Results and discussion 3.1. Results from TSNA determinations The results from the determinations of TSNAs in the cigarette tobaccos from the six brands are shown in Table 2. Results from the determinations of the TSNAs in mainstream cigarette smoke under ISO and Health Canada Intensive conditions are shown in Tables 3 and 4, respectively. The data showed that decreases in tobacco TSNA levels from 2003 to 2005 ranged from around 80% for Brand A to around 60%
Table 2 Results of determinations of TSNAsa in cigarette tobacco Sample ID
Year
NNN (ng/ g)
NAT (ng/ g)
NAB (ng/ g)
NNK (ng/ g)
Total TSNA (ng/g)
A A A B B B C C C D D D E E E F F F
2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005
487 365 105 530 375 152 441 425 143 482 479 159 464 410 164 532 386 139
570 432 131 589 490 204 548 456 169 602 603 192 600 494 221 654 473 193
27.5 16.4 6.25 29.1 19.8 7.45 24.3 19.9 8.42 28.2 24.0 9.63 27.6 21.5 11.2 29.1 19.7 8.36
714 478 110 777 601 222 648 618 185 759 718 227 700 678 281 800 615 219
1799 1291 352 1925 1486 585 1661 1519 505 1871 1824 588 1792 1604 677 2015 1494 559
a NNN, N0 -nitrosonornicotine; NAT, N0 -nitrosoanatabine; NAB, N0 -nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
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Table 3 Results of determinations of TSNAsa in MSSb under ISO conditions
Table 5 Percentage reductions for TSNAsa in tobacco and smoke from 2003 to 2005
Sample ID
Year
NNN (ng/cig)
NAT (ng/cig)
NAB (ng/cig)
NNK (ng/cig)
Brand
Sampleb
NNN (%)
NAT (%)
NAB (%)
NNK (%)
A A A B B B C C C D D D E E E F F F
2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005
9.43 7.05 3.31 18.5 13.4 6.83 18.4 14.4 6.51 24.3 20.1 7.43 20.8 16.2 8.89 22.7 17.1 9.14
13.3 9.92 5.43 26.4 20.7 10.0 24.3 21.4 10.9 34.3 30.5 14.3 28.1 23.1 13.8 34.8 28.4 14.5
1.01 0.700 0.398 1.61 1.06 0.525 1.72 0.98 0.482 2.32 1.62 0.655 1.93 1.11 0.660 2.48 1.21 0.714
15.6 11.7 4.32 33.6 25.7 10.2 30.7 22.4 10.0 37.9 36.4 13.1 35.2 28.2 14.6 41.2 32.0 14.5
A A A B B B C C C D D D E E E F F F
TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT TOB ISO HC INT
78.4 64.9 70.9 71.4 63.1 66.4 67.6 64.6 64.2 67.0 69.4 68.5 64.8 57.2 63.9 73.9 59.7 71.8
77.1 59.1 66.2 65.4 62.2 63.3 69.1 54.9 61.5 68.1 58.3 62.6 63.1 50.9 57.3 70.5 58.2 67.3
77.3 60.4 53.3 74.4 67.4 57.0 65.4 72.0 51.9 65.8 71.7 70.2 59.6 65.7 51.5 71.2 71.2 67.8
84.6 72.2 76.3 71.4 69.5 69.6 71.4 67.4 68.4 70.1 65.4 67.8 59.9 58.5 58.3 72.7 64.9 68.0
a NNN, nitrosonornicotine; NAT, nitrosoanatabine; NAB, nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. b MSS, mainstream cigarette smoke.
Table 4 Results of determinations of TSNAsa in MSSb under HC INTc conditions Sample ID
Year
NNN (ng/cig)
NAT (ng/cig)
NAB (ng/cig)
NNK (ng/cig)
A A A B B B C C C D D D E E E F F F
2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005 2003 2004 2005
34.6 27.5 10.1 47.6 33.9 16.0 36.1 30.8 12.9 52.1 40.8 16.4 38.6 31.6 13.9 47.2 32.9 13.3
54.2 41.9 18.3 70.2 51.5 25.8 57.0 42.3 22.0 75.7 62.7 28.3 58.7 46.9 25.1 73.4 50.6 24.0
2.71 1.81 1.26 3.57 2.21 1.54 2.76 2.26 1.33 4.26 2.86 1.27 3.02 2.23 1.47 4.03 2.31 1.30
60.6 45.3 14.4 78.4 62.9 23.8 63.3 51.4 20.0 84.5 76.8 27.2 69.5 59.7 29.0 81.8 61.2 26.2
a
NNN, nitrosonornicotine; NAT, nitrosoanatabine; NAB, nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. b MSS, mainstream cigarette smoke. c HC INT, Health Canada Intensive smoking conditions.
for Brand E (Table 5). The data presented by Marchand and coworkers indicated that TSNA levels in Canadian cigarette products decreased about 40% from 2003 to 2004 (Marchand et al., 2005). Another conclusion that can be drawn from the data in Table 5 is that the reductions in MSS TSNA deliveries were reflective of the reductions observed for tobacco TSNAs. However, the percentage reductions observed for MSS TSNA deliveries were generally less than those observed for the tobacco TSNAs. This phenomenon was also noted by Marchand et al. (2005). In addition, they reported that transfer rates for the TSNA were less than 10% so even a large change in tobacco concentration is not reflected fully in mainstream deliveries. There is no reason to believe that the percentage reductions in MSS TSNA deliveries between 2003 and 2005 product should be any different for ISO smoking conditions than they are for Health Canada Intensive smoking conditions. No significant changes were found in the other MSS analytes that were determined. This was not unexpected since the difference in curing was not expected to change the tobacco significantly other than to reduce the TSNA levels. Thus, the results of these
a NNN, nitrosonornicotine; NAT, nitrosoanatabine; NAB, nitrosoanabasine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. b TOB, tobacco taken from cigarettes; ISO, ISO smoking conditions; HC INT, Health Canada Intensive smoking conditions.
determinations are not included in this article. Very recently, Hayes and co-workers reported that experimental cigarettes made with indirect-fired flue-cured tobaccos gave significantly lower mainstream smoke acetone deliveries (353.9 ± 5.2 lg/cig versus 368.9 ± 7.3 lg/cig) and significantly higher smoke nicotine deliveries (2.35 ± 0.08 mg/cig versus 2.21 ± 0.01 mg/cig) than did cigarettes prepared with direct-fired flue-cured tobaccos (Hayes et al., 2007). Unfortunately, those authors did not provide analytical data on the cigarettes and tobaccos taken from those cigarettes. On the basis of the available information, we do not believe the differences in MSS nicotine and MSS acetone reported by Hayes and co-workers indicated changes in product chemistry caused by indirect-fired barns other than the reduction in TSNA concentrations. 3.2. Discussion of results In 1990, Fischer and co-workers reported on the tobacco TSNA contents and MSS smoke yields (ISO) for twenty-five brands styles of Canadian cigarettes (Fischer et al., 1990). They reported tobacco TSNA levels as follows: NNN, 256–979 ng/cig; NNK, 465–878 ng/ cig; and NAB+NAT, 572–1033 ng/cig. Assuming the cigarettes Fischer and co-workers analyzed contained 750 mg of tobacco, the TSNA levels they reported would be similar to those reported by Marchand and co-workers for cigarettes produced in 2000 (Marchand et al., 2005). More recently, Morin and co-workers reported that when they cured Canadian tobacco (1999 crop) under direct-fired conditions, total TSNA levels approached 2 ppm (2000 ng/g), but when tobaccos from the same lots were cured using indirect heating, total TSNA levels were on the order of 0.25–0.35 ppm (Morin et al., 2004). Our results indicated that total TSNA levels ranged from 1.7 to 2.0 ppm for 2003 products and 0.35 to 0.68 ppm for 2005 products. Those levels are on the same order of magnitude that Joza and co-workers reported for the tobaccos taken from cigarettes obtained in 1970 (average of 1.12 ppm) and maintained in secure storage (Joza et al., 2000). Thus, the return to indirect-firing has reduced TSNA levels to about the same levels found in product made in 1970. Based on the report by Boyette and Hamm on US-grown fluecured tobaccos (2001), lower levels would have been expected even though the US had been known for having high TSNA levels in its flue-cured tobaccos (d’Andres et al., 2003). This raises the question of other factors that might need to be considered in pro-
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ducing Canadian-style cigarettes with even lower TSNA levels. Other factors suggested have included microbial populations on the leaf (Morin et al., 2004), barn rot (Spurr and Hamm, 2003), and nitrite levels in the tobacco (Wanfeng and Yuanying, 2002). More recently, there has been a presentation on the effects of TSNA formation during the manufacture of reconstituted tobaccos (Rigoulay et al., 2006) and another presentation showing how temperature and added nitrite and nitrate increase TSNA levels in fluecured tobaccos (Fannin et al., 2006). Very recently, mainstream smoke nitric oxide concentrations have been correlated with TSNA deliveries (Hyodo et al., 2007). Taken altogether, the information in from those sources would indicate that the use of stem and/or reconstituted tobaccos in flue-cured blends could increase the TSNA concentrations above those found in the tobaccos sampled right after curing in indirect-fired barns. Continual efforts should be made to reduce TSNAs concentrations to toxicologically insignificant levels, if possible. Flue-cured tobaccos have been found with less than 30 ng/g of each TSNA and less than 100 ng/g total TSNA (Renaud and Zuber, 1996). The factors in addition to use of indirect heating for the curing need to be identified and reduced to practice, so that the concentrations of TSNAs in leaf, blend, and smoke can be minimized.
4. Conclusions We have documented the TSNA levels in the mainstream smoke from commercial cigarettes picked-up in the Canadian marketplace in 2003, 2004, and 2005. We have also documented the TSNA levels in the tobaccos taken from the same populations of cigarettes used for the smoke determinations. The decreases in MSS TSNA smoke deliveries followed the decreases in tobacco TSNA concentrations, which, in turn, were caused by the change from direct-fired to indirect-fired curing of the tobacco. However, the percentage decreases in MSS TSNA deliveries were not as great as the percentage decreases for the corresponding tobaccos. This finding shows the need for further work on understanding the relationships among tobacco TSNA concentrations after curing, tobacco processing during cigarette manufacture, and MSS TSNA deliveries. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yrtph.2008.04.009. References Baker, R.R., 1999. Smoke chemistry. In: Davis, D.L., Nielsen, M.T. (Eds.), Tobacco Production, Chemistry, and Technology. Blackwell Science Inc, Malden, MA, pp. 425–427. Boyette, M.D., Hamm, L.A., 2001. Results of the Year 2000 TSNA sampling program in flue-cured tobacco. Rec. Adv. Tob. Sci. 27, 17–22. British Columbia, 2007. What is in Cigarettes? Ministry of Health, Government of British Columbia. http://www.healthservices.gov.bc.ca/cgi-bin/ttdr_brand_ search.cgi?constituent = all (accessed February 23, 2008). Brunnemann, K.D., Hoffmann, D., 1991. Analytical studies on N-nitrosamines in tobacco and tobacco smoke. Rec. Adv. Tob. Sci. 17, 71–112. Chamberlain, W.J., Baker, J.L., Chortyk, O.T., Stephenson, M.G., 1986. Studies on the reduction of nitrosamines in tobacco. Tob. Sci. 30, 81–82. d’Andres, S., Boudoux, R., Renaud, J.-M., Zuber, J., 2003. TSNA levels in the mainstream smoke of simplified blend prototypes. Beitr. Tabakforsch. Int. 20, 331–340. Fannin, F.F., Li, X., Burton, H.R., D’Angelo, E., Bush, L., 2006. TSNA changes with cured powdered tobaccos. 60th Tobacco Science Research Conference, Montreal, Canada, September 17–20, Paper #39. Fischer, S., Castonguay, A., Kaiserman, M., Spiegelhalder, B., Preussmann, R., 1990. Tobacco-specific nitrosamines in Canadian cigarettes. Cancer Res. Lin. Oncol. 116, 563–568. Gray, N., Boyle, P., 2004. The case of the disappearing nitrosamines: a potentially global phenomenon. Tob. Control 13, 13–16.
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