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-5- v s Lmilar. For the VirK£nia tobacco samples the 8Kreement baleen run I ~i~d run 2 is sood ho~mver for the Burley $mnples it is less so. The ,heart values quoted in the table are used in the follo~ng discussion. NNN ~as found Ln all the non-cured freeze-dried smaples, a fact ~nich is st variance ~r~th the results reported by Hoffmann (&). Ie is ~ossible that NNN could be forwed between the t~ne of post-harvest ~ceatment and the time of mLalys~s. However the £reaze-drying yes L,~tended to prevent th~8 and even the first samples anAIysed conCa~ned measurable quancit£as of N~q. The curir~ processes used for the sreenhouse Scorn smnples result ~ increases in the ~ content of the tobaccos. However from the I Lmiced data obtained ~t is impossible to conclude whether the alternative ~'~rins methods result In differ@no Increases in ~ content. F f aid-Crown Samples The results relac£n8 to the field 8rows samples are siren 4n table 2. In all, aisht s~ples (Burley and Virjini8 at two fertilisstion levels and each being supplied in the cured and non-~ured forms) were saalysed uslnK both the methods ~e£erred to above and described Ln the appendix. Comparison of the result8 of the cvo methods does not imply qF ~MN foruac£on occurred as an artefact of method 1. Accordingly the results of both methods have been used and the mean values are quoted in t be Cable. In |eneral the Burlay tobacco samples contain more NNN than the comparable Virsin~a tobaccos, the one ~eeptLon to this i8 the non-~ured VirKLnia tobacco s~le ~rown unde~ normal n~t~oSen fert~l£sat£ou. This z ssult however may be anomalous because the Virsln~a s~nple concerned contains more NN~ than expected on the basis of the other V~rKin~a results. O% C~ B~,~ Co LTD - MINNESOTA TOBACCO LITIGATION

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-6- All the freeze-dried non-cured samples contain NNN - a resulc in asreaent with the results of the greenhouse Krown samples but, as noted earlier, not in agreement u~ch Hoffmann's results. However, it must be scared, chat a fairly lengthy period of rime did elapse beC~eeu~frteze- drTin8 and analysis for these samples and thls may have resulted in formation. The curins processes used for the field-grown samples were those normally adopted, flue-curing for the Virginia tobacco and air-~u~ing for the Burley tobacco. Inspection of the mean values of NNN content (Table 2) shows that there is no evidence to sugSest that curing results in an increase of NNN content. Furthermore comparison o£ individual results of the freeze-dried non-cured ~th the comparable cured smaples results in the same conclusion being drawn because only three o£ the possible eight comparisons show an increase in NI~ content on curlns. Nevertheless the results cannot be regarded as evidence to support the alternative hypothesis: curing processes result in a decrease in NNN content. All the f£eld-srown tobaccos were cultiveted under ~ different fertilisation schemes, namely normal and high nitrogen £ertilisation. The results do not indicate that either schtaue influences the ~ content of the tohacco. This aspect will be dlscussed more fully below in conjunctLon with the results on leaf nitrate content. C. r eenhou sa-~rown/F ie I d-Crown Smiles It is unreasonable to discuss in Sreat derail the differences between the twv groups og samples because they were Szown at di~.ferent t~s, under different conditions and they were kept for different periods of rime before analysis. However, the values of NNN leaf ~, [ Co LTD - MINNESOTA TOBACCO LITIGATION 0 CXD

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-7- content are 8~£1sr for both series of sa=ples, for example the mean value of 2.6 US/8 (Burley, freese-drled, non-cured, sreenhouse-~rown) should be compared with the values of 1.7 and 2.5 ~K/K (Burley, freeze- dried, non-cured, field grown at normal and hash n~crosen ferc£~sation respect£vely), and the moan value of 3.6 US/8 (Burley, a/r-cured, Kretnhouse S=own) should be compared wi~h ~he values 4.4 and 2.0 ~K/K (Burley, air- cured, field Stow: at normal and h~sh a/cross: ferciliaatlon respectively). Similar comparisons for the VirBinia samples ind£cate chat f~ld Stow: samples (uncured or flue-cured) have sl~Kh:ly h£Kher values than their Brae:house-,town counterparts. H£:ra:e Co:cent of Leaf The nitrate nitrogen analysis results are given in Table 3. He n~trite hi:raKe: was found in any of the smnplis. As expected the n£traCe values for the Burls7 :obaccos are h~Eher tha~ those of :he Virginla tobaccos. Inspection of :he resul:a for ~he field Kroum samples shows that for three out of :he four pairs of samples hilh nitro&an fercilisa:ion does result in a hasher leaf ui:raCe nicroIen consent than :ha low hi:rosen fertil£sation. The one psi= of samples for which :b/s is not the case is the Burley cured samples for which :he hi:race hi:rosen ~ontent of the leaf is ~ar~ally hisher for the normal nitroaen fert£1~sation than for the hash nicToKen fere£11saClon. While: chls is not expected it is non unreasonable to have ~solatad pairs of samples thee analyse in chls gUT because ~Lnor differences £n soil eond~:Lo~e or dra/naSe conditions could JJ~luance chase analytical values. On.,. Plottlnl :he nitrate uitroKen levels skeins: the ~ values shows a well-defined relationship for the VirKinia Breenhouee smzplas (Inset, ~ ~ [ Co LTD - MINNESOTA TOBACCO LITIGATION m O

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-8- Figure 1), high nitrate levels corresponding to high NNN levels. The relationships for the ocher groups of samples (Virsin£a. field grown; Burley, grsenhouse Erown; Burley, field grown) are less well defined Ab- although when all the samples are considered together a general trend of increas£nK NNN content with increasing u~trate nitrogen levels is apparent. Nicotine Content of Leaf The nicotine analysis are given in Table 3 and are plotted agalnsc NRN values in Yilure 2. In senate1 the results show chat the higher the nicotine content of the tobacco the greater the ~ content. This is in accord with the results of Hoffmann (5) indicatinE that nicotine is an important precursor of NNN. CONCLUSIONS In broad ca:ms the results reported here are in aSreement with those Co be found in the literature. However one important difference is apparent and it casts some doubt upon the results reported by Hoffmann. In particular, whereas Hoffmann et el. have not detected MNN in uncured tobaccos, it has been detected in all the uncured ~ples examined in this investisation. Furthermore it should be noted that the amounts found in the uncured samples in the course of this work are far hiKher qr than the detection Limit quoted by Hoffmann. Such doubts relatinK to the validlCy of the results quoted by Roffmann are £n accord rich previously reported doubts concernins the precision of hie dare.nations (7). The results of these investiKations show: 1. NNN is present in both cured and uncured tobaccos. 98" b 2, The curin$ process may be responsible for an increase in NNN content in some types of tobacco. B ~, I Co LTD - MINNESOTA TOBACCO LITIGATION

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-9- 3o ~o Virginia tobaccos contain less NNN than Burley tobaccos. The ~MN content ~s related to boCh the ultrate and nicotine content of ~he leaf. ~ ~umcgs ~. Druckrey, H. and Preu88mann, l., Natuz~rLasen., 1962, 49, 498. !. Klus. H. and Kuhn, H., Fechl. l~Ct. 8s~. Tabakregie. 1973, 1_~4~ 231. 3. Hof~nn, D., Hechc, S.S., Ornaf, R.H. and b~nder, E.L., Science, 1974, 186, 265. ~. Hoffmnn, D., Hecht, S.S., Ornaf, R.M., ~hlnder, E.L. and Tso, T.C., Y.nteruaC£onal &Sency for Kesearch on Cancer Science Publ£cacion, 1976, 14, 307. 9. Becht, S.S., Chert, C.B., Orna£, K.H., Jacobo, E., AdAu~, J.D. and Hoffmann, D., J. Org. Ch4m., 1978, 43 (1), 72. 5. Hecht, S.S., Chen, C.B., lliro~a, N., Ort~af, R.M., T8o, T.C. and Hoffmann, D., J. t~at1. Cancer Inst., 1978, 60 (4), 819. ~. BAT Report No. KD.1265 Bestricced, 13.10.75. 8. BAT Report No. L.511 Restricted, 25.2.76. 9. ~LT Report ~o. L.541 lestrictad, 20.12.76. ~ 10. Hofi~a~n, D., U-thkamp, G., and Lin, Y.Y. Internst£onsl A~eney for leseer¢h on Cancer Sc£c, ace Publication, 1973, ~, 159. B~I ~o LTD- MINNESOTA TOBACCO LITIGATION