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Y N-wi_trosariines in the Human Respiratory' Environment CO';;FTDEtiTIAL . A Method f©r th Deterinina.'~ion of Traces of N-Nitrosamines 4 y V,: Dietrich Hoffmann, Ph.D. Dzv. Environmental To xico? ogy Air.eri:can HeaT th ,F oundati;on 180' Eas t Ehd Avenue New York, N.'Y. 10026

Introduc'-3.o:1. .~ Extensive laboratory studies throughout the world have ' demonstrated that a large number of N-nitrosamines (RrRl=N-X0)' is mutagenic and ca'rcinogeniW to the experimental animal (,1-, 2) ,: . .., _ ~ .. .. . . .. . . .. 4. ' ' " -~`~' . ~~ ' ,- y . However, at present we are lacking direct human eviderice that nitrosamines are carc'inogenic to man. -hevertheless, the strong carca:r.ogenicity of dzethy].nitrosa., ine to primai:es and the possibility that these agents are pres~en it in personal pollution (4,5), urban air pollution (6)', and in food ('1.,T), available which permits the determination of.trace amounts'oL make it most desirGble to have a reproducible analytical method -9 th.,ese~ agents (ng=).0 g) in the environment. The published methods -6 permit only the detection of microgzams (10 g,)' of N-nitrosamines;; which are formed during collection by artifacts (4', 5, 8, 9).. furthermo-re, they are not reproducible' and include N-nitrosamines In general, carcinogenic N-nitrosamines are not released, directly into the respiratory environment, but are formed during .the burning of organic matter and are then released into the environment in the combustion products. Basically, there are several possibilities for the for.ma- tion of N-nitrosamines with the following three mechanisms as- the most likely ones.. 1. 2,4X R, R1=N,-i+NO+NO2, 11 R F1=NH+H:~i0 . , 2 1' 1 - 11 ' Ir . R, R =iv H 0N0 tT- ~. ?R, R1=N-NO+H2O R, R1=N-N0;-H~O' ~ R r R~ l=N-:r10+Rl10H

all three cases, secondary a^ines (R',Rl = Nii)' react either with' nitrogen oride ('I)~, nitrous acid (I2I) ,'Qr alkyl ' Althouqh most natural Products which are burned least traces of secondary acnines, they contain only any, af N02 and R-ONO'„ since the burning zones of' organic matter are reduc'ing atmospheres, and the thermic ~ traces of NOa, react as scaver:ge.: s with O, H-radicaLs . absence of N02 and R=OnO, X-nitrosamines cannot be formed combustion materials. Hozuever, cornbustion products are immediately diluted by air when they leave the combustion zone. This, then, makes it likely that part of the NO, when it leaves the areal react directly with NO,and secondary amines to N-nitrosamines (1). with eieVated temperatures,, is oxidized to NO2. This NO2 can half oy' the N0' to NQ2 , ci gare tte smoke increases signif icant].y with. paper. Since it takes about 5,10 ' minutes for the oxidation of already diluted with air which diffuses through the cigaa.ette Unaged smoke when it leaves the mouthpiece of' a cigarette contains hardly any NO2. During puffing, however, the smoke is for the determination of N-nitrosamines.in the envifonment must aging its botential to form N-n;:t; osamines. Therefore, all methods excl.ude the artificial formation of N°nitrosamines during sampling., 11. Suggested Method ~ . O ~ . ~ • h+r N The environmental agent which is most likely to.contain ~4 . traces of Nrnitrosamines is cigarette smoke In order. 7

o exclude the artificial formation of N-na.trosafi;ines diurin the trapping of the mainstream smoke, but nevertheleb7s have the potential to detect nanograms of these agents in personal pollu_ .t.entative].y decided on the f ollowing proceCure for Cigarettes are smoked under standard conditions with the full automatic 20 channel Phipps & Bird machine ('4, 10). The ,., 2U NaOH. After the second puff is taken, the trapping system is mains tream smoke is led through two g,as wash bottles f i:7.led' with .immediately purged with nitrogen for a few seconds. After 50 cigarettes are s-moked, d'zmethylnitrosamine-l2C is added as internal standard to the NaOil solution. The latter is transferred,into a flask, acidified with E-,Cl, saturated with iJaC'1 and distilled under reduced nress:ar The distillate is saturated with NaCl,, made alkaline with 2N NaOH and distilled under reduced pressure. The distillate is extracted with etr.erl dried, and the solventt is cGutiously evaporated by dryfreezing. The residue is chro;nato- and, reduced with 'LiAl H to i;n . sy;.~ae tric hydrazines and rea:cted' ., . graphed on alumi'na, the fractions withIT-activity are conlbined witrh pentafluoropropionic anhydri:de (,2FPA). aiQGs613 _x2o 0 These components with the highly negative -C0'-CF2-CF3 group can be (',R„R1'=1y--NOI 4a' ~,R,Rl~Iwi-NHI ~'~~,R',R1=N-NH-C-CF~-C)f3i-CF'13-CF2- COOH) in an elec4ron capture detector (detection limit 10-10 - 10-9' g)'. Q11~ ~ se;,~a;rated.by gas chromatogiraphy. They induce a very higih response This method'.will be at first refined for volatile N-nitrosa- mines. It will be applied' to the analysis of mainstream and saa.de-'' , strEa,m smoke of cigarettes of' a popular U. S'. brand

wi-ch high nitrate and hig%alkaloid conteni•, hig4 in n1trGte and ,., low in alkaloid coraent, and~igarettes lowin , nitrate, as w~e~ l as alkaloid content. The method wxyll be also applied fo thQ.*. 5. Magiee~~,, P~~.N. : and B:arne~~s~,, J'.N+L. :Carcinogenic Nitroso Compounds. Cancer Res. 9': 163-2'46, 1966. Druckrey, H., Preussmann, R., Ivankovic,'S., and'Schmahl, D Organotrope carcinogene Wirkungen bei 65 verschiedenen monkeys given Nrnitrosodiethyla:,iine. Proc. Am. Assoc. O'Gara, R.W., and Kelly, M.G.: Induction of hepatomas in; 1~:03'-201, 1967, N-Nit-roso-Verbd:nd'ung,en an RD-Ratten. Z. Xrebsforsch~, 69 s Cancer Res. 6: 50, 1965':. Studies in Experimental Carcinogenesis." Academic Press, Wynder, E. L. ,-and Hoffmann,. D. :"Tobacco and Tobacco Smoke. 1967, New York, 730' pp. ungen ~~ im Taba~krauc~n~.~ ~ Experientia 21:~ 4~0~~0-4'0~4,~ 19~67'.~. Neurath, G.: Zur Frage des Vorx~.om,mens von N-Nitrosoverblind- 6'. Hoffmann, D. and Wynder, E.L.: Chemical Analysis and. Carcil'iogenic Bioassays of Organic Particulate Matter.. "Air P'o:Llu:4ion" 2nd Ed., Vol., 2, Ed. A. C'S tern„ Academic Press„ New York, N'..Y'., •1968'', 187-247'. 7. Hedler, L. and Nax;guardt, P.: Occurrence of diethylnitrosa3nine Fooa' Cos-met. Toxicol. 6: 341-3'4 8, .

,w , , . Serfonte:.n, W.J., and Hurter, P.: Nitrosamines as environ ~ mental carcinogens. I.i. Evidence. fior tne~ presence of ~ ni:trosamines in tobacco smoke'condensate. Cancer Res. 26 -575-579, 1966. t Johnson, D.E., NSi11ar, J.D., and Rhoades, J.W.:. Nitrosamines in tobacco smoke. . Natl. Cancer Inst. 1vionogr..2'8':.18!1-189,. M 19'68. 1© PillsburyR 'H.C'., Bright, C.C., O'Connor, H.J.,. and Irish, r'.W.: Tar and rnicotine in c:~r,,arette srioke, J. Assoc., Offic. Agr.,C~h~emi~sts~~ 52:~~ 45~~8'r-46~~2'~, 1969. _ . ~. 11. Masuda, Y. and ::offman n, D.: A method for the determination of primary anines of polynuclear ar.o:natip hydrocarbons. J', Chro. atr5. Scy. 7: 694-6'9 , 1969. ~4, V .4

nown as strong A large number of'• N-nitrosamlnes are k carcinogens to the experimental animal including primates.. Although~we are lacking human data which discri:rninGte :I-nitrosa- mines as carcinogens to man,, an analytical method is urgently ; needled which permits the quantitative clet:er-unination of' N-nitrosa- mines in trace amounts (101) in the environment. The method mus t exclude the artificial formation of 'N-:iitrosamines du--ing A technique is proposed which should meet these two essential requirenents. The technique will be at first tested for volatile N-nitrosamines in the mainstream and side st=ea~n smoke of'various types of cigarettes and in.poilluted urban air.

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