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S ., '1 . The Less Harmful Cigarette and Tobacco Smoke Flavors - EDMOND J. LAVOIE, STEPHEN S. HECHT, DIETRICH HOFFMANN, and ERNST L. WYNDER Division of Environmental Carcinogenesis Naylor Dana Institute for Disease Prevention American Health Foundation Valhalla, New York 10595 Reports on the adverse effects of ciaarette smoking on human health in the early 1950s and the subsequent reports by the Royal College of Physicians in 1962 and by the Surgeon General of the U.S. Public Health Service in 1964 led to increased efforts by private and public agencies to discourage cigarette smoking. As a result, over 30 million people in the U.S. have discontinued their smoking habits. However, 54 million men, women, and teenagers in the U'.S. were still smoking cigarettes in 1978 (American Cancer Society 1978), compared to an estimated 64 millioni in 1963. It is unlikely that large numbers of these smokers will stop smoking soon. Therefore, efforts towards further refinement of the less harmful cigarette should be continued to reduce effec- tively the disease risks associated with cigarette smoking. THE LESS HARMFUL CIGARETTE: 1978-79 Epidemiological studies have documented a dose-response relationship of the number of cigarettes smoke& and the development of cancer of the lung, oral cavity, larynx, esophagus, pancreas, bladder, and kidney: Bioassays have also demonstrated dose-response relationships for tar applied to the skin of mice and the incidence of skin tumors, as well as for tumor development in the larynx of Syrian golden hamsters, upon daily exposure to cigarette smoke over a period of 18 months. Thus, the first approacK towards the less harmful cigarette was the reduc- tion of the tar content of cigarettes. In the U.S. the sales-weighted amount of tar in cigarettes felli from, 39 mg in 1959 to 1'6 mg in 1977 (Fig. 1). The nicotine values declined from 2.5 mg in 1959 to 1J mg in 1977. In other countries, especially in the United Kingdomy Canada, Austria, and the Federal Republic of Germany, there were similar reductions of tar and' nicotine. The gradual reduction of tar and nicotine was accompanie& by selective reductions of certainiother smoke constituents, such as benzo[a]pyrene (B[a]P) (Hoffmann et al. 1980a): 251

252/ E. LaVoie et al. 30 © e E 20 a A ca r I0 5 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 Figure 1 Sales-weighted average tar delivery of U.S. filter an&nonfilter cigarettes from 1959-1978. (Data from Wynder and Ste(Iman~ 1979.) Several developments have led to these reductions. One of the major factors was the increased consumer acceptance of filter-tipped cigarettes. Nine- teen percent of the American cigarettes were filter tipped in 1956, and in 1977, filter-tipped brands amounted to 90% of all cigarettes sold in the U.S. Major changes occurred also in the composition of the cigarette filler. These major modifications and their effects on smoke composition and on tumorigenicity of the smoke in experimental settings are listed in Table 1(Gori 1976; Wynder et al. 1976; Wynder and Hoffmann 1979). We have also found indications that the turnorigenicity of the tar, measured by the incidence of tumors on the skin of mice, has been selectively reduced since 1954.or 1955 (Wynder and Hoffmann 1979). The greatest changes in the composition of commercially blended ciga- rettes in many Western countries were brought about by the use of tobaccos which yield less tar (from new cultivars and because of more careful selection), the use of tobacco stems, reconstituted tobacco sheets, expanded tobacco lamina and stems, and tobacco leaves with better combustibility. The reduction of tar and nicotine in smoke during the last two decades has been paralleled by a significant reduction in the concentration of carbon monoxide (CO)~ in the smoke of commercial cigarettes (Weber 1976). Studies from Germany, the United Kingdom, and the United States have demonstrated that conventional, nonperforated, filter cigarettes can even deliver smoke with somewhat higher CO concentrations than do some nonfilter cigarettes (Wald . I _y r

Tobacco Smoke Flavors / 253 Table 1 Relative Effectiveness of Techniques Used Commercially in the U.S. for Reducing the Biological Activity of Cigarette Smoke (1979-1980) Smoke constituents Biological activity' Methods tar nicotine B[;3]P carcinogenicity tumor promotion Agricultural aspects Tobacco type (bright-burley) +° + . + New cultivars + + + Nitrate fertilization + + + Tobacco processing ± ± Cut ±d Use of stems + + + RTS-nonpaper processR + + + RTS-paper process ++ + + Expanded tobacco + ++ + Expanded stems + + + + Cigarette production Paper porosity + + + CellWose acetate filters' + + + Charcoal filters'' + + + Perforated filters + + + + + + + + ?~ + ? ±?C ? ++r ++ 'Comparison of gram-to-gram tar in mouse skin tests andlor hamster smoke inhalations. °Signiticant reductions. °Unknown reductions. °Insignificant reductions. `Questionable reductions. rGreater than 50% reduction. •Some reconstituted tobacco sheets (RTS) produce high levels of CO:. "Reductions of tar„nicotine, and B[a ]P (and other nonvolatiles) and volatile N-nitrosamines are, in general, somewhat greater with cellulose acetate fibers than with charcoal filters. 1976; Weber 1!976; Hoffmann et aL 1980a). This effect has been counteracted by utilization of perforated filter tips that produce smoke CO levels signifi- cantly below those of other types of cigarettes (Table 2). Such perforated filter tips, which work basically on an air dilution prineiple, appeared on the market only in recent years. It is expected that in 1979 close to 25% of all, cigarettes sold in the U.S. market will have perforated filter tips. It has recently been shown that the beneficial effects of these low-CO, low-tar, and low-nicotine cigarettes are only partially negated by more intensive smoking (Sutton et a). 1978).

t 254!'E. LaVoie et al. Table 2 Carbon Monoxide in Smoke of Cigarettes Carbon monoxide (mgfcigt) US (190% of average 1977-78 sales)a UK (1975)" Germany (1975) Getmany (1978) nonfilter regular perforated filter filter 11.6-17.0 14.4-20.0 2.8-12.8 (N = 8)`' (N = 23) (N = 9) 9-16 13-18 - (N = 9) (N = 10) 16-21 15:5-22.5 - (N = 7) (N = 17) 14.5-19.9 8.6-18.5 2.2-13.8 (N = 16) (N = 15) (N' = 9) • Average values for nonfilter cigarettes, 14.9 mg; for regular filter cigarettes, 17:1 mg;, for perforated filter cigarettes, 8.9 mg. b Average values for nonfilter cigarettes, 12.5 mg; for filtercigarettes, 16.1 mg. `N'= number of commercial cigarettes tested! TOBACCO SMOKE FLAVOR The devel'epment of the low-tar, low-nicotine cigarette required cigarette fillers with a potential for smoke flavor contribution to make these cigarettes acceptable to consumers. Such products can be realized either by selecting tobaccos rich in flavor or by addition of tobacco extracts or certain plant extracts, addition of synthetic flavor compounds, or a combination of several of these factors. Products with tobacco blends that are rich in flavor components or contain added extracts, require thorough evaluation of the biological'activities of their smoke. New cigarettes should be assayed for toxicity and tumocibenicity, so that the reduction of toxic and tumotigenic effects in the smoke of low-tar, low-nicotine cigarettes is not' offset by the introduction of unknown factors. The relationship of specific chemical smoke components with the aroma of. tobacco and its smoking quality has been the subject of extensive review (Leffingwell et al. 1972; Leffingwell 1976). In 1936, it was shown that higher sugar content an& lower a-amino nitrogen and total nitrogen are correlated with better smoking grades of flue-cured tobacco (Darkis et al', 1936). Since the levels of free amino acids and redbcing sugars in tobacco are known to affect the quality of tobacco, factors influencing their formation have been extensively studied. The inflhences of genotype, maturity, stalk position, harvesting, and curing practices have been reviewed (Tso 1972; Hamilton 1974). The enzymatic hydrolysis of leaf protein to free amino acids and the enzymatic hydrolysis of starches to reducing sugars are among the major factors

. 'A Tobacco Smoke Flavors 1255 affecting tobacco flavor. The formation of Amadori compounds by the reaction of amino acids with reducing sugars in the leaf has been shown to be extensive in flue-cured tobacco (Fig. 2). The formation of flavor compounds in tobacco and smoke occurs via Maillard and Strecker reactions. Two pathways of the Maillard reaction that directly involve reducing sugars are illustrated in Figure 2. The amine in this reaction need not necessarily be an amino acid. The presence of amines and free ammonia in smoke or tobacco indicates the likelihood that all of these substances also interact with~the reducing sugar. The Strecker reaction converts a-amino acids to aldehydes or ketones. Condensa- tion of the resulting amino-carbonyl compounds has been shown to lead to various mixtures of pyrazines (Fig. 3). Thus, these nonenzymatic browning reactions of amino acids and sugars are generally accepted as the major routes by which natural flavors in tobacco are produced. The oxidative degradation of terpenoids and carotenoids has also been shown to generate numerous flavor compounds (Enzell 1976). Additional contributors to the overall flavor and aroma of tobacco are derived from the lower-molecular-weight carboxylic acids. xuRC^ts,ax iaa ~ .aW RqilO G Fauctoee CH%ON R ~= Iql12C 0 C-Cy-R -9. C: NIK CNn R 2R-e-cu2R R' N R- -C-C6tR Amw ka0 V H011C_CHON' R C{\ WK2C fp M Figure 2 Mechanistic pathways of the Maillard~ reaction with the reducing sugar, fructose 0 ::.

256! E. LaVoie et al. 0 H 0 A. CH3-C-CHO + H2N=CI COZH ~ CHj-C-CH~N-CH-COZH ---~ I 0 M A R cH3-c-CHZNH2 + CHICHo 11. NZN1`1 CH' 4 CH2 A CH3 & I H? -, C p \CH3 N -H7 s 3 `\ ' . Hj N , ` ty3 Figure 3 Formation of a-aminoketones from a-dicarbonyl compounds and their role in the formation of pyrazines BIOASSAYS OF TOBACCO FLAVOR COMPONENTS Several alkylated 2-cyclopenten-2-ol-l-ones, whi& are known flavorants, were detected in the biologically active portion of the weakly acidic fraction of cigarette smoke (Hecht et al. 1975; S. Hecht et al., in prep.). In view of the tumor-promoting activity and' cocarcinogenic activity of the weakly acidic fraction, 3-methyl-2-cyclopenten-2-ol-l-one was bioassaye&on mouse skin as a tumor promoter. However, this compound failed to show promoter activity. _ ; Studies on its cocarcinogenic activity are still in progress. Its inactivity as a t mutagen in the Ames assay has also been recently demonstrated (Bjeidanes and Chew 1979). Maltol, a structurally related tobacco flavorant, was found to be mutagenic towards Salmonella typhimurium strain TA 100 at high doses. Assays for mutagenic activity of the distillates obtained from tobacco have recently been employed as a general screen for flavor components that may possess tumorigenic activity. Since at least 75% of all known carcinogens are active as mutagens in the Salmonella/mammalian microsomal assay system; this bioassay was employed as a guide for fractionation of the distillate. The apparatus used in this study is illustrated in Figure 4. A 1-liter flask, which contained 50 g of finely ground tobacco, was suspended in the gas chromato- graph so as not to be in contact with any side of the oven. Two special traps,' cooled in ice-water and in dry-ice and acetone were used to collect the distillate. A third trap was filled with toluene as a gas scrubber. A stream of helium (50 ml/min) was emplbyed before and during the distillation. The oven of the gas ehromatograph allowed for controlled, even heating of the tobacco. This is in contrast to pyrolysis units in which contact' with the heating coils can cause excessive and uncontrolled: localized heating effects or hot spots.

V V Tobacco Smoke Flavors !'257 t TRAP I! ICE + H20 Figure 4 Gas chromatograph equipped for collection of tobacco distillates t f TRAPIL TOLUENE DRY ICE + ACETONE The distillate in each trap was extracted with methylene chloride, dried using Na:_SO;, and concentrated to a residue by careful evaporation of solvent. In most instances, significant mutagenic activity was rarely observed for distil- lates of either trap at a dose of 1.0 mg/plate when the tobacco was heated below 250°C. Whem various groun& tobaccos were heated to 300°C, mutagenic activ- ity could be detected in both the ice-water and dry-ice and acetone traps. Although mutagenic activity was observed in the presence of liver homogenate with both TA98 and TA 100, tester strain TA98 was generally more sensitive. Fractionation of larger quantities of the combined! distillates into acidic, basic, and neutraU fractions was performed as outlined in Figure 5. Assays on the mutagenicity of these fractions demonstrated that almost all of the activity could be concentrated in the basic ether-soluble fraction. Column chromatography of the basic fraction using Silicar CC-7 further concentrated the mutagenic activity in the benzene and benzene-ethyl acetate subfractions. The mutagenic activity of these subfractions was further concen- trated by high-pressure liquid chromatography (HPLC) using a 50-cm What- man Magnum 9/ODS reverse phase column. Thus, the mutagenic, basic, ether- soluble portion of the distillate obtained at 300°C was concentrated into three fractions as shown in Figure 6. These fractions are currently being subjecte& to gas chromatographic mass spectral analysis (GC-MS). Among the more suit-

0 2581 E. LaVoie et al. TOBACCO DISTILLATE (56/I KC.OF TOBACC01 Figure 5 Fractionation of tobacco distillate into acidic, basic, and neutral 'components able columns for analysis of these active fractions are 6% Dexsil-300 (12 feet) and 3% OV-1 (6 feet) on Chromosorb WHP 80/100. Among, the compounds that have been tentatively identified were trimethylpyridine, methylnicotinate, harmane, norharmane, methyllmidazole, and diphenylpyridine. Further frac- tionation by HPLC and analysis of mutagenic activity will be required for the ultimate identification of the major mutagenic components found in tobacco distillates. Upon identification of the structure of mutagenic agents, methods for their quantitative assessment in distillates of tobacco as well as in smoke will be established. Subsequently, a qualitative an& quantitative comparison of cigarettes with extreme differences in tar and nicotine yield will be made and mutagenic compounds that are potentially carcinogenic, cocarcinogenic, or tumor-promoting will' be subjected to other appropriate bioassays. This com- prehensive approach would assure a complete evaluation of the low-tar, low- nicotine cigarette in respect to flavor compounds, an aspect of tobacco resear& that has thus far been neglected. SUMMARY Tobacco selections advanced techniques of smoke filtrationy use of reconsti- tuted tobaccos, and other technological innovations have contributed towards the development of cigarettes with reduced tar and nicotine levels in the smoke. V V

Tobacco Smoke Flavors J'259 ETHER SOLUBLE BASICFRACTION (13G/I.OKG OF106ACC0) 50% C6H6 C6H6/ EtOAc t - L 1) / 40% H2P MeOH '_ 0 40% H20/ IAPOH 125 MG SILICAR CC-7; 50 GRAMS 5% 20% EtOAc MeOH/EIOAc 6hOH/EIOAc I _ _I _ t MeOH 2) 3) REVERSE PHASE HP 89MG WHATMAN MAGNUM ODS COLUMN 4) LC 9/50 M 5) 6) eOH 1 2 3 4 5 6 7 8 9 10 1 l.-,.rJ II . . 1 -j B .,. . C. REVERSE PHASE HPl.C WHATMAN MAGNUM 9/50 CM ODS COLUMN _i__ i 0 I 2 3 4 5 6 7 8 9 10 i ' MeOH I,1 Figure 6 Fractionation scheme fon the concentration of mutagenic principle in the ether-soluble basic fraction These modifications have also diminished the smoke flavor of cigarettes, which is now increasingly compensated by tobacco selection an& the addition of organic flavor extracts or synthetic flavorants: Therefore, monitoring of the biological activities of smoke from cigarettes with added flavorants is required. The use of mutagenicity bioassays on a distillate of tobacco blends is proposed as an initial screening technique for the potential' biological activities of tobacco flavorants. ACKNOWLEDGMENTS We wish to thank Steven Carmella and' Alok Govil for their excellent technical assistance. This study was supported by Public Health Service contract NO1- CP-55666 and American Cancer Society grant BC-56. REFERENCES ~ American Cancer Society. 1978. "A national dilemma: Cigarette smoking or the fieafth of Americans. National Commission on Smoking and Public Policy. New York. ~ .~r

2601'E. LaVoie et al. Bjeldanes, L.F. and H. Chew. 1979. Mutagenicity of 1,2-dicarbonyl compounds: Maltol,, kojic acid, diacetyl and relnted substances. Mutat Res. 67:367. Darkis, F.R., L.F. Dixon, F.A. Wolf, and P.M. Gross. 1936. Correlation between composition and stalk position of tobacco produced under varying weather condi- tions. Ind: Eng. Chem 28:1214. Enzell; C. R. 1976. Terpenoid components of leaf and their relationship to smoking quality and aroma. In Recent advances in tobacco science, vol. 2, p. 32.30111 Tobacco Chemists Research Conference, Montreal. Gori, G.B. 1976. Low risk cigarettes: A prescription. Science 194:1243. Hamilton, J.L. 1974. Changes during curing of burley tobacco, Ph.D: disscrt3tion, University of Kentucky. Xerox University Microfilms,. Ann Arbor, Michigan. Hecht, S.S., R.L. Thome, R.R. Maronpot, and D. Hoffmann. 1975. A study of tobacco carcinogenesis. XIII. Tumor~promoting subfractions of the weakly acidic fraction. J. Natl. Cancer Inst. 55:1329. Hoffmann, D., T. C. Tso, and G. B. Gori. 1980a: The less harmful cigarette. Prev. Med. (in press). Hoffmann, D., S.S. Hecht, 1. Schmeltz„ E. LaVoie, and E. L. Wynder. 1980b. Recent studies in tobacco carcinogenesis: Chemistry; bioassay, and bioassay monitoring. (In press) Leffingweil, J.C. 1976. Nitrogen components of leaf and their relationship to smoking quality and aroma. In Recent advances in tobacco science, vol. 2, p. 1. 30th Tobacco Chemists Research Conference, Montreal. Leffingwell, J.C., H.J. Young and E. Bernasek. 1972. Tobacco flavoring.jor snfoking products. R.J. Reynolds Tobacco Co., Winston-Salem, North~Carolina: Sutton, S.R., C. Feyerabend, P. V. Cole, and M.A. RusselL 197& Adjustment of smokers to dilution of tobacco smoke by ventilated cigarette holders. Clin. Pharmacol: Ther. 24:395. Tso, T.C. 1972. Physiology and biochemistry of tobacco plants. Dowden, Htitchinson4 and Ross„ Inc., Stroudsburg, Pennsylvania. Wald, N.J. 1976. Mortality from lung cancer and coronary heart disease in relation to changes in smoking habit. Lancet 1:136. Weber, K.H. 1976. Recent changes in tobacco products and Iheir acceptance by the consumer. In Proceedings of the Sixth International Tobacco Science Congress, p. 47. Tokyo, Japan. Wynder, E.L. and D. Hoffmann. 1979. Tobacco and health: A societal challenge. N. EngL. J. Med. 300:894. Wynder, E. L. and S.D. Stellman. 1979. Impact of ]bng-term filter cigarette usage on lung and larynx cancer risk: A case control study. J. Natl: Cancer Inst. 62:471. Wynder, E.L., D. Hoffmann, and G.B. Gori. 1976. Smoking and health~I. Modifying the risk for the smoker. In Proceedings of the Third iVorld'Conference on Smoking and' Health. DHEW publication number (NIH) 76-1221. Government Printing Office, Washington, D:C: ~,