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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 cigarette 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 million 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 CtGARETTE: 1978-79 Epidemiological studies have documented a dose-response relationship of the number of cigarettes smoked 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 approach 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 fell from 39 mg in 1959 to 16 mg in 1977 (Fig. 1). The nicotine values declined from 2.5 mg in 1959 to 1.1 mg in 1977. In' other countries, especially' in the Uhited Kingdom, Canada, Austria, and the Federal Republic of Germany, there were similar reductions of tar and nicotine. The gradual reductioni of tar and nicotine was accompanied by selective reductions of certain other smoke constituents, such.as benzo[a]pyrene (B[a]P) (Hoffmann et a]. 1980a): 251

W I A a ® t3 0 252 / E. LaVoie et al. 25~ L_ L__L__ i NON-FILTER CIGARETTES jALL CIGARETTES ~ FlITER C1GAP.ETT£S r 35 25 10 s 59 60 61 62 63 64 6S 66 67 68 69 70 7t 72 73 74 75 76 77 78 Figure 1 Sales-weighted average tar delivery of U.S. filter and nonfilter cigarettes from 1959-1978, (I)ata from Wynder and Steilman 1979.) Several developments have le& 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 amounte&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 tumorigenicity 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 commercialiy blended ciga- rettes in many Western countries were brought about by the use of tobaccos which yield less tar (from new cultivars an& 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 e z a~, ~~. 0 0". Table 1 Relative Effectivet for Reducing the 1' Methods Agricultural aspects Tobacco type (bright-burley) New cultiivars Nitrate fertili'eatiot Tobacco processing Cut Use of stems RTS-nonpaper pro RTS-paper proces! Expanded~ tobacco Expanded'stems Cigarette production Paper porosity Cellulose aeetaEe filters" Charcoal filters" Perforated filters 'Comparison of gn "Signitlcant'reducti °Unknown reductic °Insignificant redua •Questionabie redu 'Grcater than 5o9r ; RSome reconsti lut.'( °Reductions of tar,, in general~ soinew (V N 197b;1Veber 1976; ~j by utilization of pe, N cantly below those c ~ tips, which work ba ~ only in recent years ~ sold in the U.S. m: ~ shown that the bene cigarettes are only 1 197a).

Tobacco Smoke Flavors 1'253 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 Bla]P carcinogenicity - tumor promotion Agricultural aspects Tobacco type (bright-burley) +b + + + + New cultivars + + + ?O Nitrate fertilization + + + + ? Tobacco processing Cut ±a ± ± ±?r ? Use of stems + + + ++r ++ RTS-nonpaper processR + + + ++ ± RTS-paper process ++ + + ++ + Expanded tobacco +. ++ + +?' + Expanded stems + + + + + + ? Cigarette production Paper porosity + + + ~ ? Cellulose acetate filters^ + + + ± ± Charcoal filters" + + + ± ± Perforated filters ++ ++ + + -_* ? 'Comparison of gram-to-gram rtar in mouse skin tests and/or hamster smoke inhalations: °Significant reductions. °Unknown reductions. °Insignificant reductions. °Questionable reductions. rGreater than 509o reduction. 9Some reconstituted tobacco sheets (RTS) produce high ltvels of CO_.. 'Reductions of tar, nicotine, and B[a]P (and other nonvolatil2s) and volatile N-nitrosamines are, in general„somewhat greater with cellulose acetate fibers than with charcoal filters. 1976; Weber 1976; 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 (Tabll' 2). Such perforated filter tips, which work basically on an air dilution principle, 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 al, 1978 ) L

254/ E LaVoie et ak Table 2 Carbon Monoxide in Smoke of Cigarettes Carbon monoxide (mglcigt) regular perforated' nonfilter filter filter US (190% of average 1977-78 sales)a 11.6-17.0 (N = 8)C 141.4'-20.0 (N = 23) 2.8-12.8' (N = 9) UK (1975)'' 9-16 13-18 (N=9), (N=10) Germany (1975) 16-21 15.5-22.5 (N = 7) (N = 17) Germany (1978) 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 eigarettes; 8.9 mg. ° Average values for nonfilter eigarettes.,12.5 mg; for filter cigarettes, 16.1 mg. `N = number of commercial cigarettes tested. TOBACCO SMOKE FLAVOR The development 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 tumorigenicity, soo that the reduction of toxic and tumorigenic 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 and 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 reducing, sugars in tobacco are known to affect the quality of tobacco, factors influencing their formation have been extensively studied. The influences 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 N affecting tobacco flavor. T of amino acids with reduci in flue-cured tobacco (Fib, andi smoke occurs via Ma Maillard reaction that dire. 2. The amine im this rea presence of amines and likelihood that all of these Strecker reaetion converts tion of the resulting amir various mixtures of pyraz reactions of amino acids a by which natural flavors ir terpenoids and carotenoid compounds (Enzell 1976) aroma of tobacco are dt acids. / _1laH xa~t~ a # ' ravnos[ ~ lvi~_M x v F~ ,tqYR xa~~~ ~ txn R Figure 2 Mechanistic pathways of the Mi

Tobacco Smpke 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 im 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 thata2l of'these substances also interact with the reducing sugar. The Strecker reaction converts a-amisid 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 nonenzymatie' browning reactions of amino acids and sugars are generally accepted as the majot 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. IqNC ~S}bN r "OCR Pol~ "°V 0 M Faucroat Aniwo IKie oy" Figure 2 Mechanistic pathways of the Maillard reaction with the reducing,sugar, fructose

2551 E. LaVoie et al. 0 H 0 A. CH3-C-CHO • HZMrC-COZH '-' CHj-C-CH+N-C,H-COZH R R B. 0 r CH3-C-CHZHHZ • CH3ft CH 3 \ ii0 C It 1~1 . q~ H3 . HJ,CH3 NH? 0 CH3 ftH2 -H.,0 CH3 CH3 ! I + C 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-o14 -ones, which 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-o1~I-one was bioassayed 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 mutagen in the Ames assay has also been recently demonstrated (Bjf ldanes and Chew 1979). Maltol, a structurally relate& tobacco flavorant, was found to be mutaaenic towards Salmonella typhimurium strain TA1o0 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~ containe& 50 g of finely ground tobacco, was suspende& in the gas chromato- graph ~o as not to be in contact' with any side of the oven. Two speciai*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 employed before and during the distillation. The oven of the gas chromatograph~ allowed for controlled, even heating of the tobacco. This is imcontrast to pyrolysis units in which contact with the heating coils can cause excessive and uncontrolled localized heating effects or hot spots. HZNN, Figure 4 . Gas ch_romatograph equipp The distillate in using Na.:SO,, and c, In most instances, sil lates of either trap at ; 25Q°C. When variow ity could be detecte Although mutagenic with both TA98 and Fractionatiom of Wgi and neutral fractions mutagenicity of the5 could be concentratec Column chroma concentrated the mu subfractions. The nn trated by high-presst man Magnum 91OD' soluble portion of th fractions as shown ir gas chromatographie

Tobacco Smoke Flavors I'257 t t T TRAP I TRAPIL TOLUENE ICE DRY ICE + + . H20 ACETONE Figure 4 Gas chromatograph equipped for collection of~tobacco distillates The distillate in each trap was extracted with methylene chloride, dried using Na._SOi, 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. When various ground 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 neutral fractions was performe& 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-ethyi acetate subfractions. The mutagenic activity of these subfractions wasl further concen- trated by high-pressure liquid chromatography (HPLC) using a 50-cm Wtiat'- 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 im Figure 6. These fractions are currently being subjected to gas chromatobraphic mass spectral analysis (GC-MS). Among the more suit-

a All ~ ~ 10 258! E. LaVoie et al. ETHER I 1.2 G ETHER Figure 5 Fractionation of tobacco distitlate 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 8011(?0, Among the compounds that have been tentatively identified were trimethylpyridine, methylnicotinate, harmane, norharmane, methylimidazole, 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 wiil'i be established. Subsequently, a qualitative and 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 research thaGhas thus far been neglected. SUMMARY Tobacco selection, advanced techniques of smoke filtration, use of reconsti, tuted tobaccos, and other technological innovations have contributed towardss the development'of cigarettes with reduced tar and nicotine levels in the smoke. T06ACC0 DlSTJLLATE (5G/iKGOFTO8ACC0) 50 %H6 ebDtSJ Ef I) aox 1 V/ MeOH ey! 0 ODSi 2) AOX H;P akOtt LeLi~.l 0 ! 2 !25 MG REVE YMAT z---- I __s Figure 6 Fractionation scheme for C These modif cations I is now increasingly organic flavor extrac biological activities c The use of mutagenii as an initial scrcenin€ flavorants. N ACKNOWLEDGMEN ~ N We wish to thark St€ ~ assistance. This stud N CP-55666 and Amer' N ~ REFERENCES M CA American Cancer Socie Americans. Nati(

1 1 ETHER SOLUBLE BASICFRACfION (13G/1.0KGOFTD8110C0) SILICAR CC-7; 50GRAMS 50% 5% 20% C6H6 C6yEtOA. EIOAc MeOH/EtOAe MeOH/E70Ac MeOH 1) 2)' 3) 4) 5) 6) REVERSE PHASE HPLC 89MG WHA7MAN MAGNUM 9/50CM 40% H2)/ 0DS COLU1ttN' MeOH i , 0 I MeOH 2 3 4 5 6 7 8 9 10 L. -..-j (1 1 .9. L :.. MG REVERSE PHASE HPLC 125 WHATMAN MAGNUM 9/50CM ODS COLUMN ~ ~} i L i L t 1 2 3 4 5 6 7 0 le 10 MeOH 11 Figure 6 Fractionation scheme for 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 and the addition of organic flavor extracts or synthetic flavorants. Therefore, monitoring of the biologieal 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 tobaccoo 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 health of Amerrcans. National Commission on Smoking and Public Policy. New York.

2601,E. LaVoie et at. Bjeldanes, L.F. an&H.,Chew. 1979. Mutagenicityof 1,2'-diearbonylicompounds Maltol, kojic acid, diacetyl and related 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. lnd: Eng. Chem 28:1214. l:nzell, C.R. 1976. Terpenoid components of leaf and their reli3tionship to smoking quality and aroma. In Recent advances in tobacco science, vol. 2, p. 32.30th 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. dissertation, 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) Leffingwell, 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 for smoking products. R.J. Reynolds Tobacco Co., Winston-Salem, North Carolina. Sutton, S:R., C. Feyerabend, P. V. Cole„and M.A. Russell. 1978. Adjustmentof 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, HutchirSson, 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 their 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 societalichallenge.. N. Engl: J.. Med. 300:894. Wynder, E.L. and S.D. Stellman_ 1979. Impact'of long-term filtercigarette usage,on lung and larynx cancer risk: A case control study. J. Natl. Cancer Inst. 62:47 1. Wynder, E. L., D. Hoffmann, and G.B. GoriL 1976. Smoking and health I. Modifying the risk for the smoker. In Proceedings of the Third fVorld Conference on Smoking and Health. DHEW publication number (NIH) 76-1221. Government Printing Office, Washington, D.C. Less HaZaCdi Theory and I Gf0 B. GORI Division of Cancer Caus~ National Cancer lnstitutf Bethesda, Maryland 200 Epidemiological studies between the amount of diseases in humans (Han 1966; Kahu 1966; Weir 1973). Two approaches smoke to which the pc smoking and manipulate cigarettes so that tlle hart Efforts to induce pe as judged by annual con~ tiomincreased by about_2 an increase of 2.8% c, reduction in the late 196 Health Service 1964). P Figure 1. The rates have annually since 1974 (Fe same period (1943-197, from over 40 mg to les action by the cigarette demand by the smoker. Figure 2. Data on the r same period are not reac took place. This discussion cc smoke emission from~ a consumer acceptance li' feasible solutions for rel; Cigarette smoke is : phase. The particulate condensed particles of p: