Tobacco Documents Online

AUTHOR , o.~ ISSD'1~e ~'~eok LAst C ~l," er: 15t.nO,~: P~/Ora~LDam No. : ~uct ions: DIgTP, I ~,.FI ION: PS rl) : AJK (L) : &AS (2) : ~@IC (1) : l~W (3) • :-In|r., 7.. & t',.~. Aust. (2) : ~';S f~) : CJP de S (l) : DGF (L) : Libcary ('-) :Fitc O.! Co.--'A"e : t L :: ~,~1 i i i Sent i' i (~) ~ I (4), C~) C: )i Da~e l~e Reca ~e"~A_'.'ned &uT.hor De% e Issuer Dam e I I ! (~) ~his pro fozma is in%ended ~o facilitate and de~.tl ~he IXr~S of G.R. & D.C. Beport~ f.-~ the vypescr!pt s~a~ until the repo~-t Is £1nally &~=~ve~ and ls~e~. The ~ £o=ma Zh~A;d be c~e~ced a% the time of the initial typing and a~c~ ~he ~J~sc~A~ (m~i maSt,mr after ~ep~z~tion) at all subsequent mta4~es. It mast be :e~t to cenT--~al filing with the )~8t~ when the Eq~ iJ issue&. I~a te 1 Wherever possible, ~ra~s for %~/ping~hould be written in~k. AlOe.~ tl~ing, ~he ~ypescri~t should no~ll~ be ~%u.-ned to the Autho:, who will subsequently obtain t.~e ~oup L~aAer'm and Immae~'e &pproval. = Ci_~culstlon should be determ/~ed ~I the Issuer. ~. The typescript must not be pm'esented for printin~untll the Zssuer h~s initi~llei • he pro fo~ m~,~nSt "~Te]Ma-e Z.~s~er". After prlntlr~, one boumd copy muJ% be renamed %0 the Issuer for final approval ~d the ~s~uer will e.n~u=e. - that one copy ks seen by ~he Manager. ~2n oo H ~ [ Co LTD - MINNESOTA TOBACCO LITIGATION

N'-NITROSORORNZCOTINE IN TOBACCO , i REPORT NO. RD.1683 RESTKZCTED 2z. s...t 979 AUTHOR: J.D. Green ISSUED BY: C.l. Ayres FROG. KEY. : 12.02.04 D r STEZBUT ION: _ nu Dr. S.J. Green Dr. I.W. flushes DT. ILA. Sanford R.M. G£bb, Esq. R.S. Wade, Esq. R,G, llicho].ls, Esq. Her: E. IP~Ct:ersh&ua Dr. F. Seehofer Mr. A.J. Krumgyz~k~ Dr. C.J.P. de S£que£rs Dr. D.G. l"elt:on Library Copy No. 1, 2, 3 It It 4 " " 5, 6 It fl 7 " " 8, 9, 10 " " 11, 12 " " 13 " " 14 " " 15 " " 16 " " 17 " " 18, 19 COPY NO. : ql~. - O ~m~ ~ ~. r Co LTD - MINNESOTA TOBACCO LITIGATION OO O',,

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rOG ICALI 46D Group Kelearch & Development Centre, Brit£sh-~erican Tobacco Co. Ltd., SO~N. 22nd ~ay 1979. N'-mI~OSONO~ICOTI~ IN TOBACCO (~port No. RD.1683 l~stricted) d~F--" m SUmdA~Y AlqO CONCLUSIONS N'-Nicrosono~£coC£ne (~) is a tobacco spec££1c u£trosmzLne. ~ecause of the potential health hazard i¢ presents as a carci~Kenlc aud~caK~Ic asent, several recent Invesc£1acions have been aimed at ,lete~in8 the factors which £n£1~nce ice I~I in tobacco. This ~eporc details the results of sxsm£nin~ both VirK~nia and Burley • tobaccos |ro',m. and cured -ndar d£fgerenC eond£tio~. The remulcm mhov that: (i) ~ i8 present iu both ~acured and c~ed tobaccos (ii) the cur~J~ process may be responsible £or an increase in content in 8o~t types of tobacco (iii) VirKinia tobaccos contaiu less ~ than Burley tobaccos (iv) the ~M content is related both co the n£¢race and nit.ins ~on¢~c of the 18af. ~lsc (ii), (iii) and (iv) alx~a vith pra~rLously publiskd ~ata, (i) co~liccs with recant results repotted by Hofgmann in ~h£eh was not detected in uncured tobacco 8umples. Our feB-its JJnply that, Ln orde¢ to ~n~a~se the 11N content o£ tobacco, invescilatlons~uld ~nclude consideration of the pre-curing stakes na~ly the Krowins and choice o£ varieties. ~,~I :o LTD - MINN~TA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-2- INTRODUCTION Many nicrosa~Lnes are known co be carcinogenic and therefore their ~dentification in tobacco and tobacco smoke is a cause for concern. For :onveniance nitrosanines may be divided into volatile and non-volatile types. Volatile nitrosaminms including di~uechylnitrosamutna, "methylathyl- ~icrosamine, diethylnltrosam£ne and n£troeopyrrolldine have been found in ~aiuscremm and sidestream ciearette smoke and some have been £ound to e Lesser extent in processed tobacco. This report is concerned with the aosC abundant tobacco-spacigic non-volatile nlCrosanL£Ue, N--nitrosonoruicocine - (NNN) and some of ~he factors which influence ~ts level in tobacco. Nitrosmnines were discovered in tobacco smoke ~ollowins the sus@estion of Druckrey and Preussmann (1) that such compounds may contribute to the :arcinogenicity of tobacco smoke. Most of the early work related to volatile nitrosam~nes until Klus and Kuhn isolated NNN from cigarette mnoke (2). Their study involved cigarettes which had been artificially enriched with not'nicotine. Hoveve~, shortly after this, Hofgumrm et el. reported finding NNN in the mainstzeam smoke of a cozmercially-available ~nf£1cared U.S. blended cigarette at the level of 137 uS/ciearette (3). In an attempt to identify the source o£ NNN in tobacco mmoke sevacal unburnt tobacco products rare examined; ~ was found at levels of becveen 2 and gO u@/g (3). The hi@her values were found in a fine-cut chevlng tobacco whereas levels of bmtween 2 and 7 u@/@ were typical of cisarecce end cigar Cohaccos. In a subsequent examination of unburnt Burley tobaccos @corn aC hi|h and lov nitro@an fertiliaatiou levels HOffl-,ann reported (&) that the NNN content o£ tobacco @town at the low nitrosen fercilisation yes approximately SOS of that grown at h£@h nlCroseu B ~I Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-3- fertilisation. Furthermore, as a result of examining the tobacco during flowering, topping, harveatinK, half-way curing and full-curing, Hoffmann suKgeeted that NNH is formed durinE the curing stages. .Only two air-cured Burley samples ware used eo establish this result and no indication of the reproducibility was liven. Some of the likely pathvay8 by which NNN could arise in tobacco have bean investigated by Hoffmann et el. (5). The reaction of nicotine with sodi~ nitrite at 25°C Kives • variety of nitroamnines in yields varying between 0.1X and 2.8Z, most of the nicotine remaining unchanged. In vitro exper~nts in which tobacco leaves were fed radio-labelled nicotine or nornicotine have indicated that nicotine is the major precursor of NNN (6). InveatiKetions at GRADC have confirmed the existence of NNN in ciKarette smoke (7), have shown that some 13-14Z og NNH is transferred to the mainatramm smoke, only vet-y minor amounts of NNN are deKraded during smoking (8) and cou~only used ciKareCte filters are unselective for removing NNN from clKarente smoke (9). The work reported here examines various samples of tobacco in order to assess the influence of tobacco type, KrowlnK conditions and the curing process on HI~ content. ;. DETAILS OF ~AI41~..ES Two series of a~eplee have been used in this work. The first series was Sreanhouse Krovn at GIJi~C and ineludeKi t~o L'ypes of ~bacco: aurley and Virsinia. S~ple8 of each type rare freeze-dried prior to curing, after £lut-cur~J and after air-curina, The second ssr£es was field Krown and supplied by North Carolina State University via Brown & B ~ r Co LTD - MINNESOTA TOBACCO LITIGATION co o',, P,o

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-4- Willismson. B~rley and Virginia tobaccos ware grown at high and normal nitrogen fercilisation levels. Freeze-dried s~,ples of the uncured, air-cured Burley and flue-cured Virginia tobaccos grown at both fertiliser levels were supplied. EXPgg]3~L~r~&L Two methods of analysis for NNN were used. Method I, which was based upon an outlined procedure of Hoffmann (10), was an adapted version of chat developed and reported in detail at G~&DC (7) for determining NNN in cigarette smoke. All the samples were examined using this method. Method 2 involved a modified aqueous tobacco extraction procedure and was used to re-sxsm/ne the field-grown samples for h~h'N content. This modified procedure was adopted because of the reported possibility of nicrosamine formation during analysis when chlorinated solvents were used in the extraction stake (4). However our results did not confirm this 8u~liestion. Flow diagrams and further details of the methods used are given in the Appendix. RESULTS AND DISCUSSZON Greenhouse-Grown S~mples The analytical results relating to the greenhouse grown samples are given in Table I and were obtained usinK Method I. All the values obtained for the ~MN contents are of the same order of ma~tude as the values quoted in the l£terat=re. 'gun l' and 'Run 2' detailed in Table I refer to analyses carried out usinK tobacco from two tobacco plants. Nevertheless, as the plants were Srovn under identica1.conditlo~ 4rod subsequently Created in comparable ways, the values obtained should be tJ~ Cr~ B ~. [ Co LTD - MINNESOTA TOBACCO LITIGATION