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-?xD"N^Tr.L N-NitrosGrair.es in the Human Respiratory Environ=,ent I. A::ethod -for the Le terraination o~ Traces oi N-:vytrosu.lines Dietrich Hoi"L:nunn, Ph.D. Div. Environr.;e..tul -oxicoiocy American Health Foundation 130 BGs t E::d :wer:ue New York, N. Y. 10028

~ . I nt~ oauct~o::. Exzensive laboratory studies throug:,out the world have demonstrated that a large number of N-nitrosalnines (R,R1=X-ti0) is mutagenic a:.d carcinogenic to the experimental animal (1, 2) . However, at present we are lacking direct human evidence that N-nitrosarain es are carcinogenic to man. Nevertheless, the strong ca:: c: nogenicity of diethyl;.itrosa: ;ine to primates (3) and the possi'aility that these agents are present in personal air pollution (4,5), urban air pollution (6) , and in food (1,7), make it ::,ost desirable to have a reproducible analytical method a vailabl.e w:1ich perllitS t:.C,' C:eteri~i..atioa of. trace aIi,ou:.ts of -9 t:.ese agents (ng=10 g) in the envi•ror.L;e7t. The published methods permit only the detection of n~icroc.'.a~ls (10 g) of :v-nitros4.aines; zurtherm ore, t:.ey are not rep-roGuci'ale and include N-nitrosamines, which are fo'L'med G'lurl.n y coi.lectio:: by art:.fc'.cts (4,5,8,9) . In general, carcinogenic ti-nitrosG:aines are not released directly lalto the respiratory enviro:..;;cnt, bu:: are formed during the burning of organic matter and are then released into the environ.ment in the combustion products. Basically, there are several possibilities for the forma- tion of N-ni::rosamines with the followi:.g three mechanisms as the most likely ones. I 2kX R R1=tia;-NO+N0 .2:R :t1=N-I~O+:i 0 O 6 . , 2 , ~ 2 1 -+ 11 R i21=N::+fiN0 rZ N0+: R1=ti- 0 . , 2 ,, , 2 , ~ iIi. Rl=tici+:211ON0 R ? N-\0R110'd ' , O~

l:. all t.:re~3 caJtrJ, seco:.G~.r~1 c..~~:..:.e s~:..:.es (R,R1 =\.Y) rec.ci i ei:.'r,er with nitro,e n oxides (1), nitrous acid (I!), or alkyl i.ltrlteS (lII) . Although most natural products Whicll are burned con -cain at least traces of secondary u.-ai:.es, they contain on ly traces, if any, of N702 and R-ONO, since the burning zones of organic matter are reducing atr;,osp:.eres, and the thermic activated traces of \02 reac~ as scavengers with C,a-radicc.ls. In the absence oL ~;02 ar.c: R-0N0, \-nitrosumi..es cannot be iorraec in co.;iaustio:. :,.aterials . 'r:oweve-r, co.;i,custion products are ir,mediately ailuted by air when t:.ey leave t'r.e co :.aus;:ion zone. This, then, makes i;t likely t:.a;. part of the \0, wi:e:n i t leaves the area elevGted tc~.i~pc~.rc.t.u::es, is O~:iGiZCG t0 ~02. T: ls N02 Ca:1 react d=rec::ly wit h'Nt7 a:.C secondary F,:.ii: C-s to i.-:7: i.rosa7'llnes (I) U:.aged s.;.oke W: en it leaves t:.e :,.outh-_)iece of a cigarette co:~tai:.s harGly any ~02. During puffing, however, t he smoke is alreac,y cilutec with air w:.ic:z diffuses through t'r.e cigarette paper. Since it takes about 5-10 minutes for the oxidation of :.alf of the NO to \02, cirarette smoke increases significantly with aging its potential to form Therefore, all methods for t..e c,eterminatio n of N-nitrosamines in the envizon:nent must exclude the artificial L ori:lat':io:1 of 'X-nitrosaaiiines Cauiiri, sc:.:~~pling. II. Suggested N,e thod The environmental ac}er.t which is most likely to contain traces of X-nitroSGna111es is cigarette smoke (1, 4, 5). In order

- J - to etic_uc.e t;ze arcificiul for :uticn of N-nitrosu~ir.es during the trapping of the mai:,streara smoke, but nevertheless have the potential to detect. r.anograrr:s of these agen;:s in personal pollu- tion, we tentatively decided on the following procedure for N-nitrosa;nines: Cigarettes are smio;ced uncier standard eondit.ions with the full automatic 20 channel Phipps & Bi:.a m:.chine (4, 10). The mainstrean smoke is led throi;.gh two gas wash bottles filled with 2N NaOH. After the second puff is taken, the trapping system is immediately purged with nitrogen for a few seconds. After 50 ciga-rettes are s::otied, di.i,et;•:ylr.itrosa.;L:.rie-` `C is added as internal standard to the NaOH solution. The la;."i,er is transferred into a flask, acidified with ::C1, saturated with ~uC; and distilled under rZdl:ceG pressure. The distillate is saturated w1.'i:h NaCl, made alkaline with 2:: and distiiled under reduced pressure. The distillate is extracted with et:.e-r, dried, and the solvent is cautiously evuporated by d-ry-freezir.g. The residue is chromato- graphed on alumina, the fractions wi th -activity are co:«bined and reduced with LiAl H 4 to :; nsy,,;e; ric hydrazines and reacted with pentaflr.o-rop-ro2io nic anhydride ;2c?A). , ~ (R, R1=N-\O ., pr o~) r_i, r`Z1=N-NH-C_CF2-Cc: J+Cr s-C~~2- C0O'ci ) _h20 0 These corapo:,ents with t he highly negative -CO-CF2-CF3 group can be sepurated by gas c: romutog-rup .y. They induce a very high response in an elec;.ron capture detector (detection limit 10-10 - 10-9 g). (1+; This method.will be at first refined for volatile N-nitrosa- C r„ines. It will be applied to the u na :ysis of mainstream a nd side- N ~ s..l^eG,1 s~i~o1CE9 of cigazei;i.es o.f apopula.: U.S. b1:.c:n4, ClguL'ottes ~ . ~ . ~

n: , , • + J_ J_ -' • 4. . ' J'i... ~. ~. g.1 ali . aloiG Co....E':.6., :a1C>i. i:. . i~c.~.G c..Q "1 f. i0:'1 ln a1%c,ioid co:.:.c:.t, i0'a in niai:l?, a s Wa 1~. ~ as alk ai.oid co:.i;en'G. The .i.etllod Will be also a~ ~11eG fo:.' ..1C'e analysis of polluted urbGn air. III References 1. Magee, P.N. and Barnes, J.M.: Carcinogenic Nit::oso Compounds. Cancer Res. 9: 163-246, 1966. 2. Druckrey, H., Preussmann, R., Ivankovic, S., and Sc .mC'-,';n1, D. : Organotrope carcir.o5ene 1•;ir:cu ngen bei 65 versc:ziec c nen -N-~il.i:oSo-Vez.7ialdl::.gen an aD-Rc:.l:~.e:1. Z. :{rebsi=orsch, 69: 103-201, 1967. 3. 0' Gara, tZ. W. , and :Celly, M. G. : Induc tlo:i of lle?ato mas in. monkeys given \-nitrosoaiet:.ylu.,,ir.e . Proc. Am. Assoc. Cancer Res. 6: 50, 1965. :4. Wynder, E.L., and Hoffmann, D.: "Tobacco and Tobacco Smoke. Studies in Experimental Carcinoge:.esis." Academic Press, 1967, New York, 730 pp. 5. Neurat:., G.. Zur Frage des Vorko.--=ne:.s von \'-vitrosoverbind- unge:z im Tabakraucn. Experientia 23: 400-404, 1967. 6. Ho: fr„a:.n, D. a..d k'ynder, E.L. : Cne:-nical Analysis and Carcinogenic Bioassays of Orga nic Particula~%-..e :atter. In "Air Pollution" 2nd Ed., Vol. 2, Ed. A.C. Stern, Academic Press, New York, :1.Y., -1968, 187-247. 7. Hedler, L. and M.arr,uazdt, P. : Occurrence of dietl-.ylnitrosaminQ in so;-,,e sa,72les of f6ood. Food Cos.r,et. Toxicol. 6: 341-343, 1968.

8. Ser~o::-cein, ti,;.J., and FAurt.er, P.. tiitrosanines as environ- mental carcinogens. II. Evidence for the presence of nitrosG:7ines in tobacco s:r,o{e condensate. Cancer Res. 26: 575-579, 1966. 9. Johnson, D.E., Millar, J.D., and Rhoades, J.W.: Nitrosamines in tobacco smoke. Natl. Cancer Inst. ll:onogr. 28: 181-189, 1968. 10.. Pillsbury, H.C., 3right, C.C., O'Connor, fi.J., and Irish, r. W. : Tar and nicotine in ci; srette sr;io::e. J. Assoc. Of.fic. Agr. C:.erais~:.s 52: 453-462, 1969. 11. lric sudc,, Y. and ::oi-fimanri, D. . r. .uet.A:od f:or ~`.he detei::T:ina'i.1on of pr=::ary a.u1.:.35 O-po1.j:.l:clec:.r a::o.i<<.t.lc hyG:.ocal'wti'ions. J. Chro: G'-.rg. Sci. 7: 694-697, 1969.

V. Su=u;:y A large number of N-nitrosamines are known as s;:ror.g ca-rcinogens to the experimental animal including pri.:,ates. Alt hough we are lacking human data which discril<<inF.',--e \-:.itrosa- A11.nes as carcinog.ens to man, an analytical method is ui.CJeni.].1 neec:ed w:.y.ch peri l.ts t he C1L;aY:tii:ative C:eter:aination of ti-nl.trosa" ~ :aines i n trace amounts (i0-5) in the envirc.:.,e.:t. :he raet hod a uSt exclude the artificial formation of \-:11trosc.:,lines during A technique is proposed which should uieet i:hese two essential reruirements. The technique will be at firs;: tested for vola tl.le N-nitrosamines in i.',^.e mainstream a:.Cd side si:---eu;1 smoke of various types of cigarettes and in polluted urban air.