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CHEMICAL STUDIES ON TOBACCO SMOKE LXVI. ASSESSMENT OF VOLATILE AND TOBACCO-SPECIFSC N-NITROSAMINES IN THE SMOKE OF SOME SELECTED WEST GERMAN CIGARETTESI. C. RQhl Abteilung fUr Toxikologie, Max-von-Pettenkofer Institut, ; Bandesgesundheitsamt, Berlin, F.R.G. John D. Adams and Dietrich Hoffmann Naylor Dana Institute for Disease Prevention American Health Foundation Valhalla, New York 10595 U.S.A. 1This study was supported by NCI Contract No.1-CP-55666 and by .the Federal Ministry for Youth, Family & Health of the Federal Republic of Germany.

(3-5). The levels of VNA in undiluted sidestream smoke (SS)' tobacco-specific nonvolatile N-nitrosamines (NVNA) in cigarette by cellulose acetate filter tips has been reported previously smoke (1,2) and their selective reduction in mainstream smoke exceed those in mainstream smoke (MS) up to several hundYed The presence of both volatile N-nitrosamines (VNA) and times. The concentration of VNA in SS, however, depends greatly ditions the total amount of NVNA generated in SS is comparable with that produced in the MS. So far, these observations were on the air flow around the burning cone,(4). Under standard con- only reported for popular U.S. cigarettes ajd xecentl,y for the VKk of same commercial cigarettes from the U.K. (6). In this study we analyzed the smoke of some West German cigarettes for VNA and NVNA. Material and Methods. smoker (H. Borgwaldt, Hamburg, Germany) with a rotating head Apparatus. For the MS analysis we used a 20-port automatic seconds (7). For the SS analysis we utilized a single-channel thus replacing the air in the trap with nitrogen every 2 where every second port is connected to a nitrogen source, smoker (H. Borgwaldt, Hamburg, Germany) and SS apparatus ad- justed to an air flow of 1500 m1/min (8). For one particular filter cigarette we studied the effects of flow rates of 250, 500, 1000 and 1,500 ml/min on the yields of VNA in the SS. - 1 -

For the VNA analysis we employed a Hewlett-Packard • - 2 - 10 Model 700 gas liquid chromatograph interfaced with a thermal energy analyzer (TEA; Thermal Electron Corp., Waltham, Mass.) and a (Hewlett-Packard Model 3380 A) Model 6000 A solvent delivery system (Waters Associates, final concentrate we used for the HPLC-separation a recording integrator. For the analysis of NVNA in the Inc., Milford, Mass.) with a Model 70-10 sample injection valve equipped with a Model 70-11 loop filler port (Rheo- terfaced (Thermo Electron Corp., Waltham, Mass.) with •-columns (Waters Associates, Inc.). The TEA with HPLC in- dyne, Berkely, Calif.) and Corasil II and 2 p-Porasii POPOP. scintillation counting in toluene with 0.5% PPO and 0_005$ instrument. A Nuclear Chicago Isocap 300 was used for N-nitrosamines with a Hewlett-Packard Model 5982 A GLC-MS and nonfilter cigarettes was also analyzed for individual as detector. In addition, smoke of one brand each of filter a Hewlett-Packard Model 3380 A recording integrator served 14 14 Reagents. I C]N-Nitrosodimethylamine ( C-NDMA) 0.48 mCi/ mmol) was employed as an internal standard for VNA and [2,-14C]N'-nitrosonornicotine (14.1 mCi/mmol; 9) for NVNA. The VNA standards were kindly provided by Dr. T. Fazio, Food'and Drug Administration, Washington, D.C. N'-Nitroso- A f +a+ `=

nornicotine (NNN), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1- buta:lone (NNK) and N'-nitrosoanatabine (NAtB) were synthesized by methods reported'in the literature (2,9,10)'. All solvents used throughout this study were free from VNA and NVNA accord- ing to GLC-TEA analysis. _ Cigarettes. All cigarettes for VNA analyses (except Cigarette B) were purchased on the open market in Berlin during 1978. Cigarette B and all other brands for the NVNA analysis were bought in Berlin in 1979. The cigarettes were stored in a humidity chamber at 22°+2° at relative humidity of 60+3% for . 24 hours and were selected to be within +20 mg of the average weight of 200 cigarettes. The cigarettes were filter cigarettes the butt length was determined by the length of the filter plus. overwrap plus 3 mm (11). [These smoking conditions are identical with those of the Deutsche Normen DIN 10240 for machine smoking of cigarettes (12)). smoked in a tobacco laboratory maintained at a relative humidity of 60+5% and at 22°+2°. Throughout the study we applied standard smoking conditions taking 1 puff/min of 2 second duration ' and 35 ml volume, and smoking to.23 mm butt length. For Mainstream Smoke Analysis. The procedures have been described in detail in earlier publications (2,4). In Figure 1, we have summarized the various steps of the analysis for VNA and in Figure 2 the procedure for NVNA.

-Sidestream Smoke Analysis. For the SS'analysis 6 cigarettes selected for weight and draw resistance, were smoked in the special apparatus (Figure 3). The analytical procedure has ' -been described earlier (4). smoke of a popular West German cigarette. In addition to the major VNA, N-nitrosodimethylamine (NDMA), N-nitrosoethylmethylamine (NEMA) and N-nitrosopyrrolidine (NPYR), GLC-MS analysis showed also the presence of N-nitroso- higher levels of NPYR than of NDMA. Generally, these data from the West German market. Unexpectedly, the MS contained cigarettes and 4 popular cellulose acetate filter cigarettes the results for NDMP:, NEMA and NPYR in MS and SS of 2 popalar nonfilter confirmed the earlier finding that the VNA are significantly in the MS of nonfilter cigarettes because of selective filtration lower in the MS of cellulose acetate filter cigarettes than a gas chromatogram of volatile N-nitrosamines

0 . •air flow rates during smoking, alters the MS/SS ratios of VNA, as presented in Table 2. The flow rate of 1,500 ml per minute through the sidestream smoke apparatus was determined to reflect the standard condition, because it yielded data for TPM, nicotine and VNA an•the mainstream smoke oocrparable to a cigarette smoking in the open air (Figure 3). In model experi-ments with 14C-labelled markers, we are currently studyi~ng the basic mechanisms that lead to the formation of VNA in MS and SS, and we explore possibilities for the reduction of VNA in SS. Figure 5 depicts a high performance liquid chromatogram of the NVNA in the MS of a West German cigarette and Table 3' lists the quantitative data calculated from the response of the thermal energy analyzer as a detector for tobacco specific N-nitrosami~nes in the MS of 6 selected West German cigarettes. It appears that these values are at about the same levels as are those for NVNA in U.S. cigarettes (2). We are currently studying the correlation of nicotine in tobacco, NOx in smoke and yield of NVNA in the mainstream smoke and we are examining various filters for their capability to selectively remove the tobacco-specific N-nitrosamines (5). . - 5 - _ YrF~Y,... /

:-Z' ON *w~ CIGARETTES1 ' Brand Type Smoke NDMA NEMA NPYR ng/cig ng/cig ng/cig A (20;1.,2) i Table 1. VOLATILE N-N'ITROSAMIINES IN MAINSTREAM SMOKE (MS) AND NF B (22;1.3) NF SIDESTREAM SMOKE (SS) OF SOME POPULAR WEST GERMAN MS 13.8 1.1 30.3 SS 514. 31. 510. MS 6.8 ND 11.5 SS 213. 15.4 281. MS 3.1 ND „ 8.4 SS 330. 13.2 296. MS 1.8 -ND 5.7 SS 558. 35. 700. MS 2.4 ND 3.1 SS 408. 20. 333. MS 2.2 ND 8.7 SS 538'. 30. 500. lAll values are averages of 2 analyses; the cigarettes were 85 mm long; numbers in parentheses represent mg values of TPM and nicotine, respectively. ND = not determined (<1 ng/cig). a V NF = nonfilter cigarette F ~ filter cigarette ® 3 e ~

Table CONCENTRATION OF THREE VOLATILE N-NITROSAMINES IN SIDESTREAM SMOKE GENERATED LTNDER DIFFERENT AIR FLOW RATESI CIGARETTE C 330 13.2 272 11.4 185 9.8 128 6.7 226 189 106 0 r ,f. Q `

NONVOLATILE N-NITROSAMINES IN MAINSTREAM SMOKE OF SOME 0.51 0.08 0.23 0.04 0.22' 0.06 0.12 0.02 0.18 0.05 0.18 0.06 i 1A11 values are averages of 2 analyses; the cigarettes were 85 mm long. NF = nonfilter cigarette F = filter cigarette •POPULAR WEST GERMAN CIGARETTES1 '(A `_

Schmeltz, I., and Hoffmann, D.: Chem. Rev. 77 (1977) 295-311. 10 Cancer Res. 39 (1979) 2505-2509. Morie, G.P., and Sloan, C.H.: Beitr. Tabakforsch. 7 (1973) 61-66. Hoffmann, D., Adams, J.D., Brunnemann, K.D., and Hecht, S.S.: Brunnemann, K.D., Yu, L., and Hoffmann D.: Cancer Res. 37 .10. Hecht, S.S., Chen, C.B., Hirota, N., Ornaf, R.M., Tso, T.C., and Hoffmann, D.: J. Natl. Cancer Inst. 60 (1978) 819-824. 11. Bates, W.W., Griffith, R.B., Harlow, H.S., Senkus, W., and Wakeham, H.: Tobacco Sci. 12 (1968) 192-196. 12. Deutsches Institut ftir Normung e.V. DIN 10240, Pa;t 1, (Febr. 1977) 3 pp. Compd. 10 (1974) 79-88. 9. Hu, M.W., Bondinell, W.E., and Hoffmann, D.: J. Labelled and Wynder, E.L.: Recent Advan. Tobacco Sci. 1 (1976) 97-122. Brunnemann, K.D., and Hoffmann, D.: Food Cosmet. Toxicol. 12 (1974) 115-124. . Hoffmann, D., Hecht, S.S., Schmeltz, I., Brunnemann, K.D., (1977) 3218-3222. P.iade, J,J., Adams, J.D., and Hoffmann, D.: Abstr. 33rd Tobaceo C11emists' Res. Conf. Lexington, . Kentucky, October 29- 31, 1979 p.31. Annual Report, 1978, Laboratory of Government Chemist, London, U.K. C= (