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WORLD HEA1i H-GRGANIZA'?'IOh:T 111MRNIATIOTHAL-E$GENCY -PC-tk~EoOEAR-CI4'`aF ~~~'CER- Proceed]Ap of a ~Wvrkrng Conference kid at _ tha PototerchinFsal L"fit'c, ToMinp3 Eitonian SSR 1-3 Oc-to6er 1975 - - EDITORS - E. A. WAi.,KERP. BOGOVSKI ~,. T~ICIU~'~ , .-ECHNI€ A.LEDI7TOR FOP, IARt; T W. DAVIS I IARC Scienll4lc Publicatioas-No.14 INTERNATIONAL AGENCY-FOR RES: ARCH ON CANbER LYON -1976 !

`I'~f~SA~•`~i~_ OF Ns'~RNICO~'i~I~ AND ;~,'I~€~'I't~~~ IN ~~'-ASEOUS = MIXTURESS -A'v-1) AQUEOUS -SOLIJU 10-NS - G.B. MURPA-i'H, A:.-DOMM & PeG. PEIN ciuding several widely-used drugs and-o-*_::er -bases, react with n{t:ite -to-- -form N-nitroso comoou?sds.- Hoffmffr.n et al. 111974) _also reported the rr-eg- in= suits of Liiipsky-S Singe; (i9?5;, who rePriYted that tertiary bases, nus SMuhr, -(? 973) aeported the oc-curren ze of- 40 -ng nitroso:?orn'-coti=e (1~3=in smo~e-originatir:g-from cigarettes con"ainzng tobaccc-pasticu'ariy rich in-nornieotin€ (1.95%X•Rmffman et_al.(i97`) found 137.1 ng/eigarette NINN in mainat3eam-smoke from aUS=blendeL tig$re_te- 05 mm). These authars- aBso discuaseu the aormarlon-of LW-from-nicotin-e in relation -tc the re- - ioids, of- course, irifluence~ -tttesr vaposr pressure and, carisequent1y-, t€a€ir access to-the ni+trosa-cing agent, nitrogen trioxide, Wh{ch is formed from the oxidizing nitr ic- oxide and -i-ts reaction yrelu_t ~ nitrogen diozM s. i~,s-a f irst approach, ~*e= c.eci~ed to use the alkaloids as free bases and according to theli vapour pressures at -ehe respectlve- tteaperatur¢s3 th-ese- part*y distribut-ce in aerosol ra_ticie,._ TMs distrEbution-o` the-$ik:6- saltg and free-bases, partiy dissoived- Inother smoke consti-tuterts and ence of MAL in- unhurned tobaccr,. We _there€ore decided to study the kinet-i: s of -the reaction- by vhich-_ ~Fi is formed from nornicntfne-and nicotine in gaseous mixtures cor.tair~s~g- - the reactants in conce-arations- on&-propartions closely a,-,,ro$chirg those- of tobacco smoke. The setting up of'such-a model system is not too diffi- cuit-with respect to nit-riR _oxi4de and oxygen:-b;t it is Almost _ fmpo$sible -_ _ to dR- so for the alkaloids, which-in the-origfe_ai smoke occur partly =qs- conditior_s rat-her favouredd the €.itressting -teaet ion. -Tt=e nicotine- involved tras specially purified and contained less than 1Q mg/kg- oorn4cQtine: - ihe_ unproEe: ted=nornicatfn-e - free base not res_arded-by- inssrsvlubil- -ity w}t€i_ other smoke conssita:ez:ts- swas nitrosatedd to about -20% afEer -fiva - minutes:- the available time under normal ssoking -co:d itions is-v~ry much shorter. = 0€- puri€ied nicotine under the -sgme conditionss only-0.g6% ~,ed ~ reacted to form NNN aft_er- i5 minutes at room temperature: during inhai€- ~- tion_aod exhalation of one smoke puff the rate -wou;si be Igss by sekeral - ~ ordera of mag,nitodc. -22i~ ~ I

At 80=-and=2200C-no-detectsble amyun*s, of- N'.a~'---were=-f Crmed~_ --This is_-_ nitrbc: oxide (gQdensteiri, i91gt-Neytath et -air1-Yand to-thg aimottt compiete- - - cteavage of nitrogen -Nriox3do, _the ;itrosating agenti at -thrise temperatures ohviousl'E du to the neoative-temperatorh caFf'€icient of the oxidation o€- and concentrations (tmeiin, i916). In aqueous soiutions- the formation of 1 was found to be highest at pH ?.gg at pH 1.5 and iowerz- end at pH 5.2 and higher, almost no remction occurred. The =eactior- rate was _€aund to he gFow~r-in s t~-huffer-than in 0.2 M euffet s©2utic~r~s_ ~iicc~ti~iey like many other tertiary mines - _ - © -(Lijins'c; & Singer, 1975), is nitrosatEd in aqueous solusio::s- but only at a rate measurable in parts per-thousand over aper{od of several hours. ~i.th longer react{-oa_times there seem to he two pH-dependexit-maxima. F:ITIROSATION OF NORNI00TI-"'~-_A_ND NIOOTIINg IN GASEOUS C13XiT.iTHES BY NITRIC OXfi,DE-Aa74 O.L 1RiEl1 - In order to araro~ck the co~€d;€io~ss of nisroze*-ien w Eich occur in _ toLacc o smoke aero€ois s an€sfiher of sy$=ems-weve deve' opLed. i~ao four-necked 540 sil-flasks, connected onn to another vith -TefIon tuhing,- were evacuated_ to= aho3St 0. i-r-m Hg.- The_ fi-rst was f{ Iied with - 11i/2 nitric: oxide -in= n4t-roger 3-_and 1-0 norr.icotirxe- or- 543 p"I 1 000 - nicotine (wizh-a nornicootine content of liess than i0-mg/i€g) wesg- injectdd_- through a septum -irt3_ the second. _ The two fiasks were-opened to each- other an-°~s air iet- i3i -bha-oulg~^-_ _the second, so th at_-$he-E3hoae system was adJusted to -etmospheri:- pressure anid to ar. ox;~ben-;,oa_ensra.ticn :of about = 1:02,-c:,rrespondi ng to-,that--of cigarette smoke. = Nornicotins $nd_ ni4rstine; as free basAs,-wsre in*3Pd~:.•pd in cou^eF= trations according €Q the{r vapour pressures at-22 atid- 8;~00; i.e., there was an excess of nitric oxide a_ 22°C and ar, excess of -tnhacc3 e?kaIaid-- at 800C; The reactions- were- foliow@d h} gas-chramatogr3phic determination of 1~'~, which- was - isolated by extreccion -ot the- ga~s mixtures with 10 m1 di- chloromethsne-at various time in*_e-wa, s, a new run =oeinQ made for each - geaanrement. --_Tg verify whether all the :~v'N was-thus-e3cttacted, sequentia-i - extractionse wQre carried ou>, whieh- revealgdthat no further WtMM wa- present-in the- exreacted systems The results are summarized in=iahles i and 2, which show that rsoraico- tine, as the free base and not hindered by 3ntersole;hility with other components, i.e.; nnder optimal conditinns for -reast_ion-in tbe gaseous phase as to the -propo-rtion of sa3-t- to free- hage and access of -€cee base to the-gas phase, reacts fairly-rapidiy in amanner- $iaijar t~?-~ither secon- dary bases. ?~icotine was also fo~~n-'~ to form NNN; hut after s5_minntes -at 2200 less than 3Z had- bee_n. nitrsaeted. _ At 80 and 220°C no NNN is formed from nicotine. -This seems to be _due- to the fact that because of the negative temperature cae€ficient of the- - I See p. -2a9; _ at t?~'z Se and 0E01 trioxi-dt ia :.oiy- ThE in cigar of snok-- of aaoc Tabl* 2: nixtu°es

~F~.4~544iU`~.~ OF '~i:COTI~ .- N[C~oTdF~ 229 -Tab'se 1. NiU-rosonarnicytine (NNN) 1'ormatio„ fpom. r-orntcotine fin gaseous mixtures of oxidizing ritrit-oxide-at 26©C - - Qeeos - there ~ioide be due fthe--- - oxidation Qf-nitric oxide aimost mo-nitragzo d-iozide- can be, formeda -PVr--- - _ thdrmo-re, uneer- these - conditions theaetrdgen-; io<:ide Whi.ch -is €c-tmed -does ui~iy.--is~ of thereae}iar. ipitrasating-agent, he: a+ise -the -eq not -comhine with the -abunda, t-nit: it oiside- to - form -n#trogent tri-oxido, the ~ -WO ¢ W02 ., NV^13 ~ _ . at -tho9e temperat;~_gs is sn_f-ted far to--the ieft. - At_ 10ocC, for instance. - and v<0~ ~ta-~h?v 0.o3~ of oitrogea uio~ade apgea9s i- h the form of nitrogen trioxide; - in tobacco smoice,- nQwever.,- the partial -p3 essu_ re of nitric oxide is on1, 0.0005 atm. These resu'_rs a.uldsugges~_ thzt-_th~ _.'o~at~ a~sa ~.~~Y 3iom aic~~ine ~ in ctgaiette -€M oke - makea_-no, real contrahc--tion to -the riitrosamirie content ~ --o: smosg-d3:ing the short time inYelved io r?he innalation- and exhaiatil:~ of a zwkC puff during smcking :_ Table 2.- lVitrasonarriootine INWN)- formatiom _ f ron nicotine in-gaseo-vs- m'rxtures _o-f-oxidizing nitric o_x.ide at 26e" - -% of-nicotine - ni trosat£.'_d, - ca : cifla+_ed- fromvapou_r- -pressa;r=e

WlRATH €T AL. VELOCITY Os _NliROSATION -O¢ NvRNlCOaiNE -1N AQUEOUS SOL3.TTI-GNg,AT DiFFgRENT ph \TALLES - $`erOr~'ntal- - In these riitrrsatioc -expe_riments 9:5 ul o-f 'f1-.2 or 1M buffer so?'ut-lon- -- rs€re- stirred with 81,1- saol (12 mg) nornicotine and 500 pm, l0_5 sl) IN so8ti:2a iiLtrite at &mbient tempeI`atLret - ;SR eslues were determined using an -c-le..trmetric pil meter equiped - with a_glass electrode, before edditiun of the nitrite solution which -censes a positive shift of about 0.2 units. - - At different time intervsls varging frtsm 5-60 uinutes; 0.5 m1 samples were taken and extracted with 0.1_ 4s di: hiaromeshane,- srd 2 l;1 were io- jected - into the po7t of sgss chromategssph. for M-detersfinetion.- -Ghs chromatographic-conditians-were as feblosrs; :'arisn-Aerogreph 1445; 3 a -glass _column, -2 ~---~_ lD; -5jv% glisr 1ii' on GaschromPOliCg, i00-120 mes?l, - column temperature, 250'C; - inje; zion port temperature, 250c)C, ff1smeQ - ionization detector temperature, 350": ;gss flow;-30 m1 nitrogea/min; reteaEion time for l`~, 6 ain~ For the kinetic evaluation, the iw9erse of the concentration of urs- reacted norni: atire is pletted- egsinst ti; e; the slope of the resulting j{ne is s-messure of the reaction rater - ResUtte Gas c.romathgrspliic messurem-ert=of rhe-N&W. formed- in-equeous solu- tions at controlled pH -val-jes, from 1.5 *o 7.33-showed the optimal pH to be 3.8 for the nitrosation of nornicotine fn 0.2 and 1- li buffer systems (Figs_ 1-3). At pH- 5.2 - snd : igher, and at-pH 1.5, slmosr no aitrosstion -- oycurred. At pH 3.8 and lower, deccm-positicn of the unstable free nitrous scid competes-with the nit--rosavion-reaction. A cens{derahl¢ inhiSitiosa of ai:rosst{9n was observed ~.*ith-1 :€-hui;ers as compared with C.-=2 P; sys- tems (Figs 1-& 2). The remarkable influende of pH on the_rate of nitr^stior af -norn4coa tine, which may be of im;ortence in the trspping of tobacco smoke- in- ~ ~ ~ - saue6us systems F_ becomes obvious when tan -o -of the ~-c curves is Ur~ plotted at different pH values (Fig. -3). i

NITi°cOS:AATI~:. :'F NO'uwIC`•-~Ta?E AN`f SYVOTsW 2 61 TIG. 1. . - PR:TROSvNCIRRICOT-ME - _(NNN) FOlw ~T:_Oft-IN - 0.2 M BLJlfFLERAT VARIOJS piq VALUES S3luSl= ar) 1 it iged Ich _*f va- I - - 0.26 d8ing- ©.22 ~ 0.2 ~-- 6.t~i~ ` sOlu- f Q.1 B PH to ~ Q M sL"is sat ian &t2 ftian 1~ ; ago- noruir3- in 20 30 40 tiLtie WRY - . 2 .T~ . ~ _.~.~f(e".: . ... „x~.... . 50 1

I 23-2 NEtJPRATH ES AL< _ : 1T j 4 ~ ~Y _ _ €I_~. 2; ~ - I ~`~ocr~~t: = ~~_I~:a- ,NNN ~ ~~1-~ iON - ri~_ 1.0 M B!!FFLERRT VARIOUS plu. VALUES 10 20- '40 40 5o _ 60 t7me (min) E f

NITP:SSAY-1 ~.~4 OF -NOR-ilCO?riW AND R:1O4,W F.G-. s. _ FFF_E~ OF pH 0N-~T~ OF - NITROSATIuN -OF NOR€rICUTlNb - 233 It

_ 4CURAfl, eT AL. _ VE-=MIZ -OFF -NIKOS6i.-dON OFbaCuTlN-B IN A Ye,VE- 0 U 9 gOLd T10Ng ATs DIFFRIgh'i` pH VALUES _ - - For comparison wi€h- the above -study, asimil=a?- one was -cerrie_ d ouL-on the_ nitroeatio_3 of aicotir, in-equeon€- golutions. 21-opeje-iment-al For 31$tr9satioEt, about 9-n:3--of (3.2 M buffer solution were stirred witfi 100 Ul cf specially purified_n3Fo[ine- ; aed 6.5 r-1 I b sodium-nizriLe solution at_ 22 or g€!0y. The pH vateaes of the sylutions were measured with an electroleetYic-pR meter equi-psed with- agl2ss e.l ectro3g. At_ the - time -intervals shown in Tables 3 and 4; 0.5 rl_ sample---- of the s-n-lutign were taken_, mada alkaline with aqueous poeassium cnrboqate solution and extr€cted wi*_h 100 ;:a dichlo3omethanE• of the-ex*ract 2 iil were taken for gas c3€ro~ts~graphic det€~#aatioo: The extraction procedure wa~ :.ar ~ed out at the.2arious p`s£ values used in tha °xperimec€t.- -ng dif€ereree due to pfi o€- the extracted solution was found,_ ' -- - Gas chgometographic conditions were $s €ollows; -Hewlctg-Packard R? 5751 g; _1.5 a glass bead zolumn; wash4d -with 1% Silar 5CP in chloro_'ormg teaperasore programme-r '~ ?2(39C, at -/~ir; i:~jertion poat= temperatnrer, 2's0oC; flame i;,nizgtion detector- te`perature, 33YOC; gas flows, 30 ml- nitrogea-!m3:1; 30 m1-hsdrogen/min= 5100 ml _a=r/ m3z:= _ puti€ication of nicotine was c$rried-out by dissolving 15 g nicotine in 1 Pi -hydrochloaic acid brought ta gR 3.fi5 -with bufEer: About 2 g=sod.ium nitrite were add¢d- and-the=sointien stirred for- 36 hoaars. -Af*9r _acidi€T-- _ ing with_14% hydrochloric scid,- the ffii_x[ore was=extiacied three times with dicniorometbsoe; - the remainder was made askalar.e-wish 50t- potaYeia,mm hy- droxide- solution and extrac-ted with ether.- After removal oa=thG ether the nicotine -was puri€iad by distillat_ion in vct... The boie{nga=point was determined as 1190C. -No traces-of no=nicotin£ egpeared ia- the gas ehroma- togb am: Peaut ts ; Ho€fmarn et al. (1975) .repor2ed- yields of 1-i% KIM $€te: =tre 1.our,- dep$ndiog ior: the puR vyeye= -however, nc-data on the purity of the nicotine weed was given. In order to avoid in*_e.r€erenc-e-from horrucc-tine almost-al;aays psesent_ fo samples of natural nicotine, it-was put-i€ied by nitroeaiion aa d-separa-- _ tiori of nornicotlne by distillation. -T-he purity - of the _resulting -n:.cotine, determined- b; gas chromatography, was -99.9w;- the final r_iopr.icotine con- - , ter.t was less than 10 mg/kg. - G.is chromatographic deasur emant of the MONforsPd at 22"; -in aqueo:is -_ solutions at measaired gR valses_ varying-f€oy. 1-.7 to 6.6 ?flcwAd= shat-aftez- only one hour at pH 1.7 and pK 2.5; 0_32 and 0.20 g/kg, respectively, of-_ the nicotine, -hsd been nitrosated;- The highest amounts measured in-the series were-obtained after eight days at pH-4:0-5.0, when 3.3 g/kg NNN were found fTable 3). W e- dif?er =Jim d - Tabi a- a n d ~

e srs .d'otinE ,reaent •e"ta= -aoa- - NITR'-~SATaS7N' OF '!NE 44) NKOTIt4E 53 5 Table 3. - Migrosotomicotin_so:wo, (g/ka ,, of n0cotine rgacteti, at difiereFt PH ±vali.es- - - -0=87 - - pH 2.-S- pH• 4 . 0 PH -4 . 2 - pH 5.0 - p8- &. & CI.10 - -8D _ _ - ~ 88 -8DI L.-2i 0 =2-8 _ - Na trare tYace- ND trace ND 0.46 trace trace-- trace ND 0.49 I.A j.4- -_ _ tra-ce 2.1 1.7 -0.49 E.2 _ 1.6 T.a trace- 2.3 2=-Y ~.~ - Table 4~ Nitraso~rnico~it~e -~~J~'_~i) frortned (% -of ~icpt#rae reacted) ~t -80°C and pN 3.82 T iFte -N;VN Im; ft J •

236 Wl,'RA-:rr; F-z AL. At longer- gea`bioF'£ -timees_ 2t :.oE:is aia~ r.~~iC, [ izr€ appear to _~re- C~ ~ maximai corregponding-io two, d;f -fferes~y paths- Of reect3on, ==howeverE *his finding was not purs;•ed further. Tl.e reaction proceedeti= f ast-e; at- A0aC: after f ivA minutes at pH 3.8 seore than 1% o€ -the nicotine had already reacted, the t ighest- vall€;es ob= - gerveo being reached after two hours when 6.49% NNN were €orm-ed O"Table 4). AOM9MGMaftS Ihis study was supported by US_Counci3 for Tobacco T Research G-raot No. 891.- REFM°..~ .BodEnsteio., e€. (19185 D:e Geschwir.3igk6it der Reakt!on- zwischan Stickoxyd und Sauerstoff. Z. Flekitmochem., 24, 183 . I i @egi Saaat canneJ_ . f rankft. toxic T py.ro1E (Croab3 been re i3uatch 4 CC~.+tent that it _ f oV{. ~ii - pie Elus, B. So Kuhn, B. (1973) Die 3estimmuog dgs Noroiko*_inaitrosamir-es it Rauc,hkondensat nornikotinreicher Zigarecten-. FachZ. Afitt. 3statr. amines - that Pr RNH2 4- 1 Tobak-regie, 14, 231-657 Lijioicsy, W. & 3inger,-G.M.- (1975) - Fomotion of nitroeamirwes from terti-rr=y tmin€s and ritrmis goid'. In: Bogavski, P. & Walker, E.A., ed., N- Aitroso-Compo-zz-16-in the -Artviran-ert,- Lyoni Ir_[e_rnatsonal Agency for Research on Cancer (IAPC Scientific PubUoationa No.9), pg= 111-114 - ~-_ the car catalyi and _tak Saaherri - alao Pr Io4ua,= 1n oeut Ueefer aairea dealkyl ~-= Gkielio (1936) - Ean&woh der or-org~:~.azhen Cletanie, B.AuE1 ~ 4Si, Wyir-.heiz i p. 738 Hoffmann, D., Hecht,-h.ti., Or-nai, R.-ni_ & Wynder, E-L_. (19,7411 V-NitEoso- -nornico=in€ Io tobacco,. - Science, 186, _26Sg426? 9offma^n, D., hachka-m; ; G. & Liu, Y.`I. ;137 5-) Che?%caf tt-.4di°s or. -baccco ar.-noke. XXV%. _On the isolation _mand i(Ursf:,ficasion of t5alriile ard? nor- voWil&e N-ritroemi_rZnea mid Yydra.z yna$ in cigewatte ~-~l:,e. Inc Bogavaki. P. & Walker,_E.A.,- ed., N.Nitrwaac, Cbwwwnde in _ the E?wirowe:}tc, Lyon, International -444mcy for Research on-^ailcer (l~?; Se_::entifica Pt:VFica= tio,`a8 No.9) 3 pp.-1S9-l`a5- -