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c 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 1,0595 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 by the Surgeon General of the U.S. Public Health Service in 1964 1962 and ~M1 , . . =F:r ° 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 tively the disease risks associated with cigarette smoking. THE LESS HARMFUL CIGARETTE: 197'8-79 ' ' Epidemiological studies have documented a dose-response relationship of the : t: .. number of cigarettes smoked and the development of cancer of the lung, oral ~.~ cavity, larynx, esophagus, pancreas, bladder, and kidney. Eioassays have also VW'4=: 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 . 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- 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 United Kingdom, Canada, Austria, and the Federal Republic of Germany, there were similar reductions of tar and nicotine. The gradual reduction of tar and nicotine was accompanied by selective reductions of certain other smoke constituents, such.as benzo[a]pyrene (B[a]P) (Hoffmann . . . :=•`-~ et al. 1980a). . 251

. 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 I(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 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 fobacco` 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 cQrtventional, nonperforated, filter cigarettes can even deliver smoke with t sornewhat higher CO concentrations than do some nonfilter cigarettes (Wald Table l ' .Relative Effecti• for Reducing tf• . Agricultural aspec : Tobacco type (bright•burle. New cultivars Nitrate fertiliza~ Tobacco processii Cut Use of stems RTS-nonpaper RTS-paper pro; Expanded tobat Expanded steni Cigarette product i' Paper porosity Cellulose aceta filters" Charcoal f Iters 'Perforated filtei -'Comparison o °Significant rec "Unknown redi °lnsignificant rt ..~~ •Questionable r rGreater than 5(1 •Some reconstit 'Reductions of in general, sor 1976; Weber 197 by utilization of cantly below tho: tips, which work only in recent ye sold in the U.S. shown that the b cigarettes are on' 1978')1.

....:-+ti~c.... .~.%~N. .~ . ••Yaww_~. .~-.,:. Methods tar nicotine BlalP carcinogenicity - tumor promotion, Agricultural aspects ...•~.,. Tobacco type (bright-burley) +" + ._`+'fijQvl i Airw cnltivara . -1- •t- ' Nitrate fertilization + Tobacco processina Cut ± ~.-,- ;:t~2. . . Use of stetns . . + + + s + + + RTS -nonpaper process RTS-paper process + + + + Expanded tobacco + . ++ + " r~' 1 Expanded stems • + + + •+ ~~ - ' - a3? Ctgarette production Cellulose acetate tilters" Charcoal filtersh + + + ' Perforated' filters " ' ' ++ ++ ++ 'Comparison of gram-to-gram tar in mouse skin tests andlor hamster smoke inhalations: "Significant reductions. QUnknown reductions. alnsignificant reductions. •Questionable reductions. . • tGreater than 50% reduction. 6Some reconstituted tobacco sheets (RTS) produce high levels of CO•:. t'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 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 (Table 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). raper poroslty

2541 E. LaVoie et al. Table 2 Carbon Monoxide in Smoke of Cigarettes Carbon monoxide (mg/cigt) nonfilter regular fiiter US (190% of average 1977-78 sales)' 11.6-17.0 (N = 8)F 14.4-20.0 (N = 23) UK (1975)" 9-16 - 13-18 (N = 9). (N = 10) , Germany (1975) 16-21 15.5-22.5 (N = 7) (N = 17) a= Getmany (1978) 14.5-19.9 8.6-18.5 (N = 16) (N = 15) perforated filter 2.8-12.8 (N = 9) 2.2-13.8 (N = 9) ' Average values for nonfilter cigarettes, 14.9 mg; for regular filter cigarettes, 17.1 mg; for perforated filter cigarettes. 8.9 mg. ',Average values for nonfilter cigarettes, 12.5 mg; for filter cigarettes, 16.1 mg. `1V = 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, so 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; Leffingweli 1976). In 1936, it was shown that-higher sugar content and lower a-amino nitrogen and totall 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. Tlte influences of genotype, maturity, stalk pdsition, 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 affecting tobacco flavor of amino acids with rec in flue-cured tobacco ( and smoke occurs via Maillard reaction that c 2. The amine in this presence of amines ai likelihood that all of tht Strecker reaction convt Iion of the resulting a various mixtures of p) reactions of amino acic by which natural flavor terpenoids and caroten compounds . (Enzell 19 aroma of tobacco are acids.: - e~cx o c cx~=e WV -0 cxo f- Mechanisde pathways of the

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 Stcecker reactions. Two pathways of the Maillard reaction that directly involve reducing sugars are illustrated in Figure ed not necessarily be an amino acid. The i on ne 2. The amine in this react free ammonia in smoke or tobacco indicates the presence of amines and likelihood that all of these substances also interact with the reducing sugar. The Con Strecker reaction converts a-amiciti ncid ~to aldehydes ketones. leadsta pon of the resulting amino-carbonyl compounds these nonenzymaue browning 3 ). , various mixtures of pyrazTnes 4Fig. reactions of amino acids and 'sugars are generally accepted as the major routes roduced. The oxidative degradation of - o are b p acc flavorsin to by which natural terpenoids and carotenoids has also been shown to generate numerous flavor l n ce compounds (Enzell 1976). Additiona lower~molecular-weight carboxylic aroma of tobacco are derived from th acids. ~_~~ -7,j Zr' ~..•~..

Figure 3 Formation of a-aminoketones from a-dicarbonyl compounds and their role in the formation of pyrazines BIOASSAYS OF TOBACCO FtAV/OR' COMPONENTS Several alkylated 2-cyclopenten-2-ol-l-ones, which are known flavorants, were detectedd 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-1-one was bioassayed on mouse skin as a tumor promoter. H'owever,, 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 (Bjeldanes and Chew 1979). Maltol, a structurally related tobacco flavorant, was found to be mutagenic towards Salmonella typhimurium strain TA100 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 i-liter flask, which contained 50 g of finely ground tobacco,, was suspended in the gas chromato- graph ~o as not to be in contact with any side of the oven. Two speciaCtraps, 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 in contrast to pyrolysis units 'tn which contact with the heating coils can cause exoessive and uncontrolled localized heating effects or hot spots. Figure 4 - Gas ch.romatograph equ The distillate using Na_S0a, an( . In most instances, Iates of either trap . 250°C. When vari ity could be dete, Although mutagen with both TA98 x . Fractionation- of la and neutral fractic mutagenicity of 11 could be concentr4 Column chroo concentrated the r subfractions. The trated by higli-prc man Magnum 9/C soluble portion of fractions as showr gas chromatograp `..~.:'.~ ..

. • Figure 4 Gas cF.romatooraph equipped for collection of tobacco distillates The distillate in each trap was extracted with methylene chloride,, dried using Na_SOa, 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 TA100, tester strain TA98 was generally more sensitive.' Fractionation of larger quantities of the combined distillates into acidic, basic, and neutral 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 91ODS 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 subjected to gas chromatographic mass spectral analysis (GG-MS). Among the more suit-

258! E. LaVoie at al. TOBACCO DISTILLATE (5G/1KGOF TOBACCO) ' s1iH6 CA s 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, barmane, 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 wili 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 1ow-tar, low nicotine cigarette in respect to flavor compounds, an aspect of tobacco research that has thus far been neglected. - • ` SUMMARY Tobacco selection, advanced techniques of smoke filtration, use of reconsti- tuted tobaccos, and other technological innovatioris have contributed towards the development of cigarettes with reduced tar and nicotine levels in the smoke. Figure 6 Fractioaation scheme i These modificatio is now increasinl organic flavor ex biological activitic The use of mutag, as an initial scrcer flavorants. : ACKk11OWLEDGM We wish to thank assistance. This s CP-55666 and Ar REFERENCES Amesican CancerSc Americans. t

Tobacco Smoke Flavors 1259 BASIC FRACTION I I3G/I.D KG OF T08AOC01 ETNER SOLUBLE SILICAR CC-7; . 50 GRAMS . 50x 5% ~}{~ EtOAc EtOAC M~/EIOAe 21 .3) 4) REVERSE PHASE HPLC 89MG WHATMAN MAGNUM 9/50 CM ODS COLUMN' MeON 8 6 7 8 9 10 ~ 4 2 3 ..It- - J l...r- ~ . g 'C`

260! E. LaVoie et al. Bjeldanes, L.F. and H. Chew. 1979. Mutagenicityof 1,2-dicarbonyl compounds: Maltol, kojic acid, diacetyl and related'substances. Meitat 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. 32.30th 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. Thorne, R.R. Maronpot, and D. Hoffmann..1975. A study of tobacco carcinogenesis. XIIL Tumor-promoting subfractions of the weakly acidic fraction. J. Natl. Cancer Inst. 55:1329. Hoffmann, D., T.C. Tso, and G. B. Gori. 1!980a. The less harmfu2 cigarette. Prev. Med. (in press). H'offmann„ D., S.S. Hecht, I. 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 IvI. A. Russell. 1978. Adjustmcnt of smokers to dilution of tobacco smoke by ventilated' cigarette holders: Clin. Pharinacol. Ther.. 24:395. Tso, T.C. 1972. Physiology and biochemistry of tobacco plants, Dowden, Hutchinson, . 2-:M~ and R s I Str ud bu P l i , rg, van os nc., o s ennsy a. Wald, N.J. 1976. Mortality from lung cancer and coronary heart' disease in relation. to ha i ki habit L 1:136 nges n smo ng . ancet c . Weber, K.H. 1976. Recent changes in tobacco products and their acceptance by the di I P & Six h I n rocee ngs of t t consumer. e nternational Tobacco Science Congress, p. Japan. .. :aw : 47. Tokyo , Wynder, E.L. and D. Hoffmann. 1979. Tobacco and health: A societal challenge. N. , Er+gl. 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. .l. 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 FVorld Conference on Smoking and Plealth. DH'EW publication number (N1H) 76-1221. Government Printing Office, p ^~ wasnington, u.t.:. G10 B. GOR! " aivision of Cancer Ca National Cancer Instit Bethesda, Maryland 2 Epidemiological studi between thc amouitt ( diseases in humans (1: 1966; Kahn 1966; W 1973). Two approact smoke to which the smoking and manipul cigarettes so that the h Efforts to induce as judged by annual c tion increased by abot an increasY of 2.8% reduction in the late I Health Service 1964) Figure 1. The rates.1t, annually since 1974 ( same period (1943-1 from over 40 mg to action by the cigaret demand by the smokc Figure 2. Data on th same period are not r took place. This discussion smoke emission fron consumer acceptance feasible solutions for Cigarztte smoke phase. The particuI: condensed particles o