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PIIILIP'MORRI'S U. S~. A. ~ INTE'R-OFFICE' CORRESPONDENCE Dwilmullu Richmond, Virginia To: Dr. A. H. Warfield Date: 2 August 1991 From: S. A. Haut Subject: Tobrscco Alkaloirls and the MS Delivery o,f'TS1V'A INTR'ODUCTION Previous:work with water-extractables depleted (WED)~ tobacco; i.e.,, water-waished (Ww) tobacco' and base web (BW)',,' has shown that removal of thee water-extractabl!es in DBC burley (leu), DBC bright (Br), and blended oriental (;Or) filler virtually eliminates the delivery of NNN andINAT into mainstream (MS) smoke by affecting both transfer and pyrosynthesis.' The portion of MS contributed by the transfer mechanism is reduced almost entirely by extraction of preformedINNN and NAT. The portion of NNN and NAT in MS contributed by p;•rosynthesis is reduced by the removal of other components that are potential precursors such as nitrate and alkaloids. MS NNK delivery is not significantly affectedieven though preformed NNK levels in filler are greatly reduced'. The specific.ro1!e of ionic nitrate has been studied in WEDifillers and'shown to be an, important factor in the MS delivery of' NNN and NiAT' but not NNK.' In the literature, tobacco alkaloids have also been studied as possible TSNA precursors but conclusions have often been contradictory.' One previous literature report has claimed that nicotine, a tertiary amine alkaloid, was the major source of MS NNN and NNK in smoke via a complex series of nitrosations and rearrangements." However, subsequent publications in,the literature and work in _roject 6908 has indicated otherwise.''' Alithoughnot a clear consensus, the secondary amine tobacco alkaloids (nornicotine, anatabine, and anabasine) appear toibe major precursors of both preformed and MS NNN, NAT, and NAB, respectively,, being formed by a one-step simple nitrosation. The formationlof preformed and MS NNK is different since.the corresponding precursor, pseudooxynicotine, has not been definitively found in tobacco' and has alhigher chemicall reactivity than the other alkaloids.10 In this report, two areas of the alkaloid/TSNA relationship that are not yet clearly understood are addressed: (1)~ are tobacco secondary minor alkaloids precursors to MS TSNA and (2) is nicotine a precursor to MS NNK (especially in WED tobaccos) and if not what is the precursor to MS NNK? N 0 EKPERIMENTAL ~ ~ Nicotin (NIC) and'anabasine (p;B) were obtained from Henry Secor in ~ Chemical Research and had been vacuum distilled and stored cold in sealed ampoules. Anatabine (AT) and pseudooxynicotine (PsON) hydrochloride were ~ ~ obtained from Dr. G. B. Neurath and used as received. ' ~ A"typical" base web (TBW) was obtained from the pilot plaint (20 May 19B6).' Burley base web (J6BKH') was from, the first crossed~-solubles base web study.' Low alkaloid burley filler (J5BUV) was.obtained by Bob Carpenter fromi Q: the Leaf Department. DBC burley (J7AAM) was obtained by Ralph Hellams. Water-washed filler was prepared'as described previously.' C91-04547

A Devilibiss Model-15 Atomizer headidtiven by house nitrogen was used to apply solutions of alkaloids onto the fillers. A minimum volume of 95% EtOH was the preferred solvent for this operatiomsince it allowed the fillers to reequilibrate quickly with little or no secondary extraction. Water was required for the addition ofnitrate. A minimum volume was used and the tobacco allowedito reequilibrate to constant weight in a conditioned room before hand- fabrication into cigarettes. Cigarette filler weights were adjusted to compensate for added components to attain the same tobacco content for a control and experimental cigarette. The nitrate source, reagent grade magnesium nitrate, Mg(NO,,)=•6H;O,` was stored in a vacuum!desicator to prevent the:accumulation of atmospheric moisture. Target nitrate levels (~expressed as nitrate nitrogen, NNI) were chosen (about 0.4%) to mimic those found in DBC Bu, a tobacco known to deliver a high proportion of pyrosynthetic TSNA into:MS smoke.' NNI analyses were performed by the Analytical Research Division. Minor alkaloid determinations were performed by Bob Levins using GC-NPD." No filler TSNA analyses were conducted on altered fillers since it was assumed that in the short term there would be no change in preformed TSNA levels by the added!components relative to controls. M'S'T'SNA data were obtained'by the usual methods using replicate 1 X 5 cigaret.te smokings with GC-TEA analysis." The GC-TEA response factor for NAT was arbitrarily assigned to NAB in the integrator calibration table to obtain relative measures of NAB. RE'SU1LT5 AND DISCUSSION Tobacco Secondary Alkaloids as T'SNA Precursors: Low alkaloid burl!ey (ILABu) is an excellent substrate for studying,pyrosynthesis because the total! alkaloid content is low (<0.01%) and the nitrate content high (NNI = 0.78%)1. Anabasine citrate was added to LABui. The resuSts are shown in Table 1. Table 1 MS T'SNA ANAIYSIS OF LOW AL~'Orn BLtRT.E~' WITH ADDED ANABA INE CITRAT " MS (,ng/cigt)~ AB % NNN NAT NAB NNK J5BUV (NNI = 0.78$') 01.003 156 136 216 75 + ainabasine 01.50 136 73 8862 58 Ninlike any of the results for nicotine, a large and significant increase ini NAB (about 340-fold) is observed., Table 2 shows the effect of,both anabasine and nitrate addition to Bui Bw,= a WED substrate. Table 2 M'S TSNA ANALYSIS OF BURLEY BASE WEB WITH ADDED ANABASINE CITRAmE pLnD NT~mRAmF" MS (ng/cigt) AB' NIiII NNN NAT' NAB NNK J63KH (NNI < 0.04%) <0.001 % <0.04 % 15 110 4' 332' + nitrate - 01.5 % 142' 42 2'0, 164 + anabasine 0.25 % - % 13 9 2'081 337 + anabasine + nitrate 0.25 %' 0.5 % 132 33 12'7819 27'6

As has been observed previously with Bu BW and TBW, addition of nitrate' alone does cause increases in the MS delivery of the TSNA (about 5-fold for NAB'). Addition of AB alone increases MS NAB delivery (about 50-fold'). This increase is substantially less in the low nitrate environment of Bu BW than the increase observed in the high nitrate environment of the LABu (Table 1). With both nitrate and alkaloid'added, the amount of MS NAB increases considerably (>3000-fold). The exact nature of'the nitrosating agent(s) in filler and'smoke is unknown but the presence of nitrate (natural or exogenous) has been associated with the formation of TSNA,` Anabasine and nitrate together cause significantly more NAB formation than the addition of either alone. Water-washed burley, like Bu,BW, is depleted!in water-extractablee components.'I Experiments which oversprayed anatabine and nitrate in a manner similar to the above experiments are shown in Table 3. Table 3 MS TSNA ANAZYSIS OF WATER-WASHED BURLEY OVERSPR AYEn. WITH ANATABINE CITRATE AND NITRATE" MS (ng/cigt) Water-Washed Bu (J7AA'M) AT NNI NNN NAT' NNK Control 0.001 % <0.04 % 17' 10 117 + nitrate - % 0.4'0 % 40, 1'6 2116. + anatabine + nitrate 0.10 % 0.410 % 51 371 24!9. Here again, addition of nitrate caused an increase in all three measured TSNAA but the greatest increase occurred for NAT upon the additionlof both AT and nitrate. Based on the above results and those of'previous literature and internal work, it is quite apparent that the natural secondary amine minor alkaloids, " nornicotine, anatabine, and anabasine are the major sources of the tobaccoo nitrosamines NNN, NAT, and NAB by a direct nitrosation reaction. Nitrosationn during curing leads to the preformed (filler) TSNA'' and nitrosation duringg smoking leads to the py,rosynthetic portion of MS TSNA.' The chemical behavior of the secondary alkaloids imthe presence and absence of nitrate may also account, at least in part, for the differences in. TSNA among bright,, burley, and oriental tobaccos. (a) Bright and burley tobacco have similar alkaloid'level's but nitrate is generally higher in burley. Preformed and MS levels of NNN and~NAT are also generally higher in burley. Some burley varieties which contain lower levels of secondary alkaloids show lower filler and smoke TSNA levels even with normal nitrate and nicotine content.'"' Also,, the same variety of burley tobacco grown in different locations shows varying nitrate, alkaloid, and TSNA content." (b) Bright is low in nitrate but, probably because of the high temperature used in flue-curing, bright tobacco filler still has preformed TSNA (although not as much as in burley which is air-cured at ambient temperature),. This preformed TSNA is reflected in MS levels due to transfer but pyrosynthesis does not occur on the same scale as in burley.' (c) Blended oriental generally contains liittle or no nitrate'°' and only slightly lower alkaloid levels than bright or burley. It also contains relatively little TSNA in either filler and smoke. Some components of the oriental blendi (e.g., Greek Katerini, Basmas and Macedbnians varieties) do contain measurable nitrate levels, have higher filler TSNA, and behave similarly to bright during smoking." Nicotine as Precursors to NNK',: For some time now, the chemical behavior of NNK has been mentioned in our reports as being consistently unlike that of NNN' and NAT. In general!, NNK has remained relatively unaffected by a variety of

chemical and physical manipulations of cigarettes anditobacco. The case of base webs and water-washed fillers has been most perplexing. TBW,' for example, has very low levels of preformed TSNA andinitrate compared to feedstock. MS dolivery of NNN and NAT is, consequently, also lower. TBW MS NNK delivery relative to preformed filler TSNA andTSNA precursors (alkaloids and nitrate) is higher than feedstock. The theory was proposed that particular pyrolytic properties of the fiber in BW caused even residual levels of an unidentified alkaloid precursor (possibly nicotine) to form NNK. Previous R&D experiments addressing this theory have shown that added nicotine or nicotine citrate do not alter the MS'TSNA deliveries of low alkaloid substrates (LTF, LABu, LABr, or blended Or) relative to controls.' These experiments were, however, conducted prior to the discovery of the WED MSNNK behavior. In order to test the above NNK hypothesis in a WED filler, an experiment identical to the previous ones was conducted. Nicotine was applied to TBW and the MS TSNA determined. The results are shown in Table 4. Table 4 MS TSNA ANATrYSIS OF BASE WEFt WITH nnnE NI OTrNE CITPtA'TE: a Filler (ng,/cigt) MS (ng/cigt) Sample NIC NNN NAT NNK NNNi NAT NNK TBW 0.06 96 29 411 19, 15 11 354 + NIC 2.3 % ---------------- 17 12 357 This result is similar to the earlier findings'" in demonstrating that residual free nicotine (i.e., free base or salt) is not the precursor to any MS TSNA (NNK in particular), from,BW and washed-fillers. Pseud=rvnicotine as PT uTsors to NNK: PsON, a secondary amine, is theoretically the immediate nor-nitroso precursor to NNK (analogous to AB and NAB). Unlike NN, AT, or AB, the presence of PsON itself has not been clearly established!in tobacco.'' The MS results of the addition of PSON (as the hydrochloride) to LABu is shown in Table 51. , Table 5 MS TSNA ANALYSIS OF LOW ALKALOID BURI,EY WITH ADDED PSEUD0O7{YNICOTINE HYDROCHL'ORID'E" MS (ng,/cigt) NNN NAT NNK J5BUV (NNI = 0.718%) 211 122 116 + PspN (1%) 236 124 911 The almost 8-fold increase in NNK indicates that, in theory, PsON can behave as a direct precursor to NNK. Whatever PsON that may be present in any tobacco material before smoking is probably formed as part of a cascade of oxidation/degradation products ofnicatine rather than as a natural biosynthetic product like nornicotine or anatabi!ne.10 Preformed NNK in normal tobacco may actually result from the nitrosation of PsONi or similar compounds formed in minute amounts as intermediates in the oxidationLdegradation ofnicotine during, curing,. Under basic pH conditions, PsON tautomerizes to N'-methylmyosmine -4-

(NMM). NMM is unstable towards degradation in air at room temperature but can be nitrosatediin solution to form NNK." In base webs and water-washed tobaccos, free PsONiis not likely the major precursor of NNK since it is, in fact, water-extractable. There are currently several working hypotheses under study to explain the origin of'NNK in these fillers (andipossibly normal tobaccos),. Briefly, they involve bound'species which are nicotine-like" (mentioned in the previous section), PsON-like, or NNK- like.'` In particular the bound NNK theory" ' is the:simplest and has the broadest application. However, none of the above, including PsON itself, can yet be clearly eliminated based on current knowledtge. CONCLUSIONS It is apparent that the secondary minor alkaloids nornicotine, anatabine,, and anabasine are the immediate precursors of NNN, NAT, and NAB, respectively, imtobacco and MS smoke. They are the major source of these three TSNA. 2') Nicotine as the free base or salt is not a direct or major precursor for NNN or NNK in normal or water-extracted tobaccos. 3) Mainstream smoke pyrosynthesis of NNNi, NAT, and NAB on the scale observed in tobacco requires both the appropriate secondary alkaloids and a source of a nitrosating,species, probably de:rivedifrom ionic nitrate. A deficiency of one of these precursors, especially a secondary alkaloid, reduces the formation of the TSNA in filler andismoke. 4): Pseudooxynicotine, if' present in tobacco, can be a source of both filler and smoke NNK. The role (if any) of the tautomer, N"-methylmyosmine, in NNKK formation is not known. The contribution to MS NNK delivery by a bound precursor seems likely but is as yet unproven. « k~k~tiId : ~ 1) Investigate varieties of tobacco which, because of naturally low levels of leaf nitrate andLor secondary minor alkaloids may contain lower levels of TSNA in filler and smoke, e.g., Maryl'and=' or American Aromati'c.. 2) Investigate the possibil!ity of selected breeding, genetic manipulation, and the like for generating tobaccos which have a low secondary alkaloid to total alkaloid ratio. 3) Design and initiate a study, independent of the bound nicotine study in ~ progress, specifically targeted towards determining the existence of a bound 0 NNK or bound NNK precursor. ~ N ACFCNOWILEDGEldENTS G? ~~i The author gratefully acknowledges the hel!pful discussions of Ray Morgan, ~A Al Warfield, Caro:lyn Keene, and Rbbin Kinser. The indispensible assistance of C Ralph~ Kaiser who fabricated and, machine-smoked all experimental cigarettes, and performed the smoke workups., is gratefully appreciated.

FOOTNOTES :,. a. Haut, S. A. TSNA Analysis of Water-washed DBC Bright Tobacco Filler. Memo to A. Hi. Warfield; 1990 January 8. b. Haut, S. A. TSNA Analysis of Water-washed DBC Burley and Blended, Oriental Tobacco:Fillers. Memo to A. H. Warfield; 1989 August 21. c. Haut, S. A. The:Effect of Washing with Aqueous Reagents on DBC Burley MS'NNK. Memo to: A. H'. Warfield; 1991 June 24. Haut, S. A.; Lambert, E. A. TSNA Content of Crossed~-Soliuble Base Web. Fillers. Special Report 88-056, 1988 November 14. '} a. Morgan, W. R. Stud'ies:of Tobacco Specific Nitrosamines Pyrosynthesis: Past, Present, and'lFuture.. 21 June 1989. Special Report 8I9-03'01. b. Morgan, W. R. Transfer versus Pyrosynthesis as a Source of TSNA in. Smoke Memo to R,. D:. Kinser; 1918'5 July 5. c. Morgan, W. R. Mod'e!of'Trans!port of TSNA into Smoke. Memo to R. D. Kinser; 19815 I July 5. 4 ) Haut, S. A. The Effect of Ionic Nitrate on the MS Delivery of'TSNA. Memoo to A. H. Warfield; 1990 November 29. See also reference 3ai. a. Djordjevic, M. V.; Gay, S. L.; Bush, L. P.; Chaplin, J.. F. Tobacco- specific Nitrosamine Accumulation and Distribution in Flue-cured Tobacco Isolines. Ji. Ag ri._. Fbod _h. m. 37 (3) : 752-756; 198'9. b. Chamberlain, W. J.; Baker, J. L.; Chortyk, O. T.; Stephenson, Mi. G.. Studies on the Reduction of Nitrosamines in Tobacco. Toba..o S_i:n.P. 30;: 81-82; 19'8'6'.August 8. c. MacKown, C. T.; Eivazi, F'.; Sims, J. L.; Bush, L. P. Tobacco-specific N-Nitrosamines: Effect of Burley Alkaloid Isolines and Nitrogen. Fertility Management. J. Agric. Fbod Chem. 32(6):~ 12'69-1272; 1986. d. Chamberlain, Severson, R. F.; Stephenson, M. G. Levels of' N-Nitrosonornicotine in Tbbaccos Grown under Varing Agronomic: Conditions. Toba --o, S' :ien_ 2'8 : 156-158 1984 April 4!. e. Mirvish, S. S.; Sams:, J.; Hecht, S. S. Kinetics of Nornicotine and Anabasine Nitrosation in Relation to NI-Nitrosonornieotine Occurrence in Tobacco and to Tobacco-induced Cancer. J. Natl. Cancer Inst. 59(4): 1211-1213; 1977'October. `) a,. Hoffmann, D.; Hecht, S. S. Nicotine-derived'N-Nitrosamines and. Tobacco-related Cancer: Cinrrent Status andiFuiture Directions. Cancer. Research. 45: : 935-944; 1985 March.

b. Hoffmann, D.; Lavoie, E. JI.; Hecht, S. S. Nicotine: A Precursor for Carcinogens. .an - eT L . .s. 26: 67-75; 19,85. c. Adams, J. D.; Lee, S. J.; Vinchkoski, N.; Castonguay, A.; Hoffmann, D. On the Formation of the Tobacco-specific Carcinogen 4-('Methylnitros- amino)-1-(',3-Pyridyl)'-1-Butanone:During Smoking,. Cancer Letters. 17: 339-346; 19'S3'. d. Hecht, S. S.; Chen, C. B.; Ornaf, R. M.; Hoffmann, D. Chemical Studies on Tobacco Smoke LVI. Tobacco Specific Nitrosamines: Origins,. Carcinogenicity, and Metabolism. rAR_ Sci.n if,_ Publications. #19: 395-413; 1978. e. Hoffmann, D.; Rathkamp, G.; Lui, Y. Y. Chemical Studies on Tobacco Smoke, XXVI. On the Isolation~and, Identification of Volatile and Non- volatile N-Nitrosamines and Hydrazines in Cigarette.Smoke. I'ARC Sc;' ...n t; ; P ub> i a ; ons. 15I9'-165, 1974. ') a. Morgan, W. R. Effect of NicotineiAddition to Filler on TSNA Formation in Mainstream Smoke. Memo to S. Tafur; 1924' June 19. b. Kinser, R. D. Effect of the Addition of Nicotine on the TSNA content of Mainstream Smoke from Low Alkaloid Tobaccos. Memo to Dr. E. B. Sandiers; 1985 June 10. c. Kinser, R. D. PM Notebook No:. 8137 and 8i8'8. d. Lambert, E. A. PM Notebook No. 8050, p. 107. e. Duplication of'Hioffmann's original work clearly reveals many experimental difficulties (especially with the radiochemical purity of his nicotine and'identification of products) associated with the claim that NNN and NNK are derivediprimarily from nicotine (personal communications with S. B. Hgssam, R. D. Kinser, and W. R. Morgan). ") Studies at R&D, typified by reference 7, have long lead us: to doubt the conclusions of'the work in reference 6. Recently, the literature has also produced papers reporting similar conclusions. a. Djiordjevic, M. V.; Desai, D:.; Sigountos, C. W.; Brunnemann, K. D.; Hoffman,, D.; Bush, L. P.; Burton, H. R. Mechanistic Studies into on the Formation of 4-(Methylnitrosamino):-4-(3-Pyridyl)butyric Acid and Other Tobacco-specific Nitrosambnes During Tobacco Processing. Paper #43, 4i4th Tobacco Chemists' Research Conference, Winston-Salem, North Ca:rolina,; 1,9'90 October 2. b. Caldwell, W. S.; Plowchalk, D. R.; deBethizy, J. D. The Nitrosation of Nicotine: A Kinetic Study. Paper #22, 44th Tobacco Chemists' Research Conference, Winston-Salem, North Carolina; 1990 October 1.. c. Fischer, S.; Spiegelhalder, B.; Preussmann, R. Investigations into the origin of tobacco-specific nitrosamines in mainstream smoke. Carcinoqenesis, 10 (5):,72'3-730; 19'90.

d. Fischer, S.; Spiegelhalder, B.; Preussmann:, R. Preformed tobacco- specific nitrosamines in tobacco-role of nitrate and!influence of' tobacco type. Carci'noqen.es,_s, 101(8): 1511-1517; 1989. e. Djordjevic, M. V.; Brunnemann, K. Di.; Hoffman, D. Identification and andianalysis of'a nicotine-derived!N-nitrosamino acid and other nitrosamino acids in tobacco. Carcinogenesis, 101(9) : 1725-17'3'1; 1989. f. Djordjevic, M. V.; Sigountos, C. W.; Brunnemann, K. D.; Hoffman, D. On the Formation of 4-(Methylnitrosamino)-4-('3'-Pyridyl)-BUtyric Acid. Paper #27, 43zd Tobacco Chemists' Research Conference, Richmond, Virginia; 1989 October 3. An intensive investigation is currently underway in the TSNA project to unequivocally determine whether PsON (or NMM) is present in cured tobacco. There is preliminary evidence by GC-MSD that suggests the presence of PsON or NMM in normal,, water-washed, and baseweb tobacco fillers (personal communications with C. K. Keene). Its presence may be natural or attributable to microbial action or air oxidation.t0 There is no literature evidence for PsON'being a natural minor alkaloid in tobacco. Most references deal with the tautomer NMM'. a. Pailer, M. Chemistry of Nicotine and Related Alkaloids (Including. Biosynthetic Aspects),. In: Von Euler, U. S. Tobacco Alkaloids andl Related ComT,ounds. New York: Pergamon Press; 1965; 15-36. b. Kuhn, H. Tobacco Alkal'loids and Their Pyrolysis Products in The Smoke., In: Von Euler, U1. S. Toba ..o Alkaloids and R-la d Compounds. New York: PergamonlPress; 1'965; 37-51. The chemistry of PsON is interwoven1with nicotine-N'-oxide and N-methylmyosmine (its tautomer) along with other similar oxidative and degradative prodiacts of nicotine. The following references are typical. a. Kisaki, T.; Koiwai, A.; Mikami, Y.; Sasaki, T.; Matsushita, H.. Transformation of Tobacco Alkaloids. Beitr. Tabakforschi. I~rnt. 9(5): 308-316; 1978 December. b. Brandange, S.; Lindblom, L.; Pilotti, A.; Rodriguez, B. Ring-Chain Tautomerism of Pseudooxynicotine and Some Other Iminium Compounds. Acta Che~L_,n Scand. 1$L. 37: 617-622; 1982. c. Uchida, S'.; Maeda;, S.; Kisaki, T. Conversion of Nicotine into Nornicotine and N-MethyImyosmine by Fungi. Agric. B'iol_ Chem. 47'(9q: 194'9-1953: 1983. d. Maeda, S.; Matsushitay Hi.; Mikami, Y.; Kisaki, T.; Structural Changes of N-Methylmyosmine Based on pH. Ar,_c. Biol. Chem. 44(7):~ 1643-1645;. 1980. e. Maeda, S.; Matsushital, H.; Mikami, Y.; Kisaki, T.; Synthesis and Characterization of' N-Methylmyosmine. .A„gric. B'iol. Chem_ 42'(11) : 21177- 2178; 1978.

f. Kisaki, T.; Maeda, S.; Sasaki, S.; Mikami, Y.; Uchida, S. Pseudooxynicotine. Japani. Kokai JP 52/134094 f7'7/'134!0941. 1977 November 9. e. Wada, E.; Yamasaki, K. Degradation of Nicotine by Soil Bacteria. Z, Amer. Chem. Soc. 76:, 155-157; 1954 January 5. f. Hain!es, P.; Eisner, A. Idbntification of Pseudooxynicotine and its Conversion: to N-Methylmyosmine. J. Amer. Chem. Soc. 72: 1719-11721; 1950 April. g. Rayburn, C. H.; Harlan, W. R.; Hanmer, H. R. Rearrangement of Nicotine Oxides. Ji. Amer. Ch m. o!. 72: 1721-1724; 1950 April. Levins, R. Capillary GC/NPD Analysis of Tobacco for Nicotine and Minor Alkaloids. Memo to S. Hasisam; 1989 June 1. a. Baker, P. G.; Ferguson, R. N. Determination of Nonvolatile Nitrosamines by a GC/TEA Procedure., I. Methods. Speciali Report 8'1-113; 1981 May 6'.. b. McKay, C. L.; Ferguson, R. Ni.; Baker, P. G. Determination of Nonvolatile Nitrosambnes by a GC/'TEA'Procedure. II. Filler and Mainstream Smoke Results. Special Report 82-168; 19,82 May 18. c. Warfield, A. H., Tafur. S. 6908 Annual Report - Chemical Studies of Condensate. Annual'. Report 83-071; 19183, April 21. d. Warfield, A. H:.; Kinser, R,. D.; Zimmermann, M. L. Modifications to the Models 6102' and 610 Thermal Eiaergy Analyzer Systems. Memo to J. L. Charles; 19i8'.4 January 2'6'. e. Kinser, R. D. Alumina Column Chromatography in Nitrosamine Analysis. Memo to E. B. Sanders; 1985 August 22. f. Morgan, W. R. Modification of TSNA Analysis Method: A'ddition of MegaboreO Capillary Column Chromatography. Memo to R. D. Kinser; 1988 January 15. ") Haut, S. A. PM Notebook No. 84!6,8', p. 1!06, 113. Haut, S. A. PM Notebook No. 8468, p. 1215. . ~ ~ ") Haut, S. A. PM Notebook No. 8468, p. 188. ~ ~ ") a. The chemical and physical properties of each alkaloid do seem to have N some influence in attenuating the respective TSNA levels found in fillerr " ~ and smoke but these are not major factors.' See also Warfield, A. H.; Hansen, K. R. Extraction of Burley andiBright Fillers: Removal of TSNA N NPh and Alkaloids. Special Report 88-058'.; 198'7 November 14.

b. There is also indirect evidence that simple derivatives of the minor alkaloids.such as the naturally occurring NI'-acyl'alkaloidls (e.g., N'-acetyl-nornicotine and'N'-octanoylnornicotine) can be precursors to TSNA." ") a. Warfieldl, A. H. 69102 ' Annual Report - Biochemical Special Investigations Annual Report 86-0I8'4; 1986 March 24. b. Warfield'i, A. H. 69,02 Annual Report - Biochemical Special Investigations Annual Report 87-0$I8'; 1987 August 7. Haut, S. A. PM1Notebook No. 8891, p. 171, 176-177, 18!0'. A memo on these experimentsy is.currently in progress. a. Personal communication with Roger Bass based on data from the Tobacco varieties Field Tests. b. Haut, S. A. Tobacco Specific Nitrosamine Content of "Low-Nitrate"' Burley Tobacco. Memo to R. D. Kinser; 1985 July 26. Personal communication with C'. K. Keene and based on a stud+y of eight oriental varieties (report in progress)'.. See also Haut, S'. A. PM Notebook 8:768, p. 31and'41. Also, Akehurst, B. C. "Tobacco." Longman Group Limited, Londony 1968. ''') Haut, S. A. PM Notebook 8768, p. 1-3, 42, and reference 19. Haut, S'. A. PM,Notebook No. 8282, p. 93. Haut, S. A. PM Notebook No. 8282, p. 114. _`) Warfield, A. H. PM Notebook No. 8196, p. 77, 9'3'.. "), a. Personal communications with W. F. Hempfling, E'. A. Lambert, and. M. H. Zoller. b. Hempfling, W. P. i7inextractediNilcotine as Potential Source of NNK in Smoke: A Proposal for Study. Memo to Dr. A. H. Warfield; 1989 April 25. Warfield, A. H.; Haut, S. A.; Keene, C. K.; Morgan, W. R. Current Theoretical Considerations omthe Origin of Pyrosynthetic NNK from Water- Washed Fillers andBase Webs. Memo to R. D. Kinser; 19!91 April 4.