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FlDENTIA ASSESSMENT OF THE CAECINCXGENIC N-NITROSODIETHATTOI.AMINE American Health Foundation Submitted' for publication. i :}•~~ : IN TOBACCO PRODUCTS ANIDiTOBACC4'J SMO1GEI .fx Klaus D. Erunnemann and Dietrich Hoffmann Division ofEnvironmental Carcinogenesis Naylor Dana Institute for Disease Prevention ,vi 3t" ' -M ~ hU''ec4, ----~~-~~ .^.~5..._.~.

5 Running Titles Nitrosodiiethanolamine imTobacco Psoduicts. ' ~^i ' , . ~ ' .. . ~ . . . . . .. . . .. ~ Sj, , ' e

f. k.~free of NDELA. The tobacco of 0'.Sg smoking products contained, M~ 'ng/cigarette, whereas hanid-suckered tobacco and its smoke were 'tained 1i 1i5-42'0 ppb of NDELA and their smoke containedi 20'-290 1115-42'01 ppb of NDELA and the mainstream smoke from such pro- 4 d~ucts yielded 10~-618 ng of NDELA per cigar or cigarette. NDELA ~ levels in, chewing tobacco, ranged from 2'20 to 280 ppb and , in 2 NDELA in tobacco is formed from the DELA residue during the to- bacco processing. Based on bioassay data from various laborar 0.6-1!.9 ppm of NDELA it is evident that the major portion of the five analyzed MH-DELA pre arations.contained between commercia~l snuff pr~o~~ducts~, were~ 3',2~~0~~0! and 6,800 ppb- w,Althiough iories which have showrn, that NDELA is a relatively strong car- cinogen and based on, the results of this study the use of' ~. _,.._ .... ' ME-DELA for the cultivation olf tobacco is questioned. .. 0 97 f f Abstract w 4 simple~,-"~repro~~du~cible~ GG'-TEA method has_been d'ev~e~loped m>~.~,F :~t~l,and tobacco smoke. .The extract of tobacco or the trapped par- for~'~th~e~~ analysis o~f~ N~-nitrosodi~ethanolamine ~ (NDELA)i i'n to~b~acco~ . ., . ''~"ticulates of' tobacco smoke are chromatog,raphed on silica gel. '='i•~`The NDELA containing fractions are concentrated,_silylated and 1v` analyzed with a mod~ified' GC-TEA system,. ; NDELA-14~ serves as ` internal standard'for the quantitative analysis. :,Experimental cigarettes made from tobacco which was treated with the sucker "ry ' g,ro th inhibitor maleic hyd!raaide-diethanolamine (MEi-DELA) _con- T(.r { ,,1 I )kr Yr WL -t r, ay.

INTRODUCTION N-Nitros©diethanolamine (NDELA) appears to be one of the 0-~%-~ "'rmost'widespread N-nitroso eompoundis in the environment of deve- loped countries (1i).'--`NDELA is found in a variety of products ' , .. . - .. . ' .. „ . . ti . . . .:,Y` ... . . . . . . .. ~ incl.udingcutting fluift'and cosmetics and is formed by reac.- `_tion of dii' . .... , . . ..:,:. ., . _ . ethanolamine and triethanolamine with N.-nitrosating agents (1-3).0 'r'Formation of NDELA is 'largely due to the exten- sive use of these two amino alcohols. =..ND'ELA induces carcinoma y liver,and kidney of ihe ,ry in rats (4-b)anal carcinoma or ttrie 11 ~nas~al,cavity~ and papillomaiof the t~rachea in S~yr~ian gol!den h~~am- toxicity" was induced in Syrian golden ham- sters compared to those receiving the smoke of'control cigaret- . ,:. es or of' cigarettes containingi 1A of the sodium salt of' MEi n the United States about 3'.2 million pounds of'maleic hydraaid'e are used annually as systemic plant growth inhibitor (12), about 40i$' as the M!H-DELA formulation (13, 14). In a preliminary study, we reported that processed OS to- bacco treated with MH-DELA contains 100-170, ppb of NDELA (15'). For the analysis, NDELA was enrichedl from tob!acco extract by sters ('7 )i .' ,`,Thie'carcinogen penetrates rat ,. skin ('9) and 10) ~~. . :.r . skin (8), and human is primarily excreted via the urinary tract.(8', n a 2'6 week inhalation study with smoke of'cigarettes -''7:co1ntaining~' 1$~~ male~~ic~~ hyd~razide~-die~th~ano~~lamine~~ (~,MF3+-DELA)~ a~ "Mild '. ? NY 1. 9W th 4: ,,, V

/ k: 0 Abbrev i~ations: So .4-a i Y~O 11 15 T. '1i •.. P.Q9w' u"t ~ A1DET,A, ' IQ-nitrosodiethanolamine; MH', maleic hydtazide; ;DELA, a3i- ,;17"ethanolamine; TEA, Thermal Energy' Analyzer; :_GC~-MS, Gas Chroma- ! ... tography-Mass-Spectrometry; BSTFA, N,O~-bis(Ttimethylsilyl)tri- _ .. . 1 ,. . . ,. ., .. , . . ; , ~ ' fluoroacetamide; TMCS, Trimethylchlorosilane; NNN, 2Ni', n~troiso- , nornic~ot~ine~~;~~ NNK, 4'~-m~,ethyl-4~-nitosamino-1i-~~(3':-pyridyl~)~- butanone;, NAT, N''-nitrosoanatabine. ,, , a, i r: L y 4 ~4W791 t;

repeated chromatographic clean-up steps fallowed by ..;gas chro- neither 1arg,e'-scale studies nor the trace analysis of NDELA in cigarette` smoke. `'".:It was the purpose of this investigation to ~~'`develop"a reprod~uciblle and simple method for the determination ~"of NDELA in processed tobacco, and in tobacco, This method, however, was cumbersome and permitted

ly1ATERTAhS' ANdD METHODs 'X L:i.'t :; i~-1rJ ... ,.,, i.. ~. .k ~Js'~" s.3f1 'C~3t'li.f 3!~ ~ For the~ analiysis of the mainstream smoke we used a 2'0i-port xc~ .,.I .: 1 .., Th Eli t o~ Cbr tw,Taltham t~ass )'tth'is was c r n ~ . J Apparatus <.i.:.;",.,? Hamburg', Germany)i with' rotating ,,l~~ead of which,, ever~r 2nd port was connected wrilth, a nitrogen ,,. , , . . ,. .- sec«~(16)."~For the sidestream smoke analysis we utilized a. s~o~iaro~,`_'thuis~~`replacing~~~ the'~~a~ir in 'the~ traps with n~itr~ogien, every ermany an ' single-channei sm~alcer (H. Borgwaldt, -Ham urg,,, .. _ b, ~ )' di sidestreamismo}~e apparatus with an' air 'f]~ow rate of 25, ml~'sec, f~.ed by removing, the TC detector and acco~o~dating the GC inter- . ~.~.r.. ..~ 1i7). '-A Hewlett-Packard Model 700 g'as chromatograp~'~~~' was modi- face face and the ceramic pyrolyaen of' the Model 543 Thermal._ Energy Ana y~er (, ermo e accomplished by drilling a'hole through the oven housing thus allowing, a direct' interface' to, the chromatographic column. The 'TEA pyrolyzer interface was further modified by addition of a :.,, ~ , reducing union (Swagelok SS'-8''10-6'-41) into which a raph'ite fer- . rule (Applied Science, State'College, PA., part #15457) was placed (Figure 1) This allows the connection' of tt'fe gas chro- mato!graphic column directly to the interface rather than to the Y` brittle ceramic tube itself. S'ilylations were carried out in, 1 ml hiypovialis ( P'ierce 12901). Mass spectral analyses were per- formed on a Hewlett-Packard Model 5710!-59'8'0 instrument. }6

Cigarettes N a.,. ,. . . , .. , ~ . The commercial tobaccolproduicts were purchased!"in 1i980 on the open market in Westchester County, N.Y'. , and one fine cut :~tj.~ ~-Otobacco was obtained in Tennessee. Experimental cigarettes made from ~~ hand-suckered tobacco, ~from, MH-DELA treat~ed~ to~lbacco~ i: andifrom hand-suckered tobacco g,rownion pesticide-free soil on ~vq Prince Edward Island, .Canada,' were kindly supplied by Dr. T.C'« Tso,' USDA, Beltsville, MD1. ;,.-.1iR1 cigarettes from the University fa.of Kentucky served as reference standards. All cigarettes and 'cigars were stored in a humidity chamber at 60i (+ 3$ ) R. fl. Throughout this study, we applied the standard smoking °s conditions for cig,arettes (18), and' cigars (19). ~..;.L;~r;:Reagents Y .z .Y.79,.t•~~'....T .. ~ NDELA was synthesized iniour laboratories by nitrosation, of d~iethanolamine and subsequent purification on basic alumina ~~(~Woe~lm~~),,` activity III;~ its p~~urity~ was a~s!sured~ by~ G~,C~-MS.~ NDELA- 14C was obtained by nitrosation of dliethanol[i2-1'4'C]I-amine HCl (spec. act. 116 mC~i/mM;~~, Dhiom, Prodlucts~,, R. Bollywoo~d,, CA) and~ purification by column chromatography using 250 g silica gel (1-34041, -Baker Chemical Co. ) and ethyl acetate. M'ethanol, ace- tone, tone, ascorbic acid andlCelite 545 were obtained from Fisher ~r = Scientific Co. Acetonitrile and BSTFA with 1% TMCS' were pur- r M chiased -at Pierce Chemical Co. The chromatographic packing OV-225 was obtained from Analabs, North Haven, CT.

,LTobacco Anialysls ~ Forty~ g~ fine~ cut~ tobacco were~~ combined~with~ ,I.O.g g.ascorbic~ 120 ng NDEII.A-14C' ( 2301, 0100 . dpm)i and 400 ml ethyl acetate. { This mixture was stirred overnight and then filterEd thxough ~~,. ..: .. . _. . ~ . . *wR..-.....,.......:....... , ...... .~....-~w-..Y~~.. ' . . ` 't ~ . . , . . ~ : . P,4,..,Celite 5451..w The filtrate was thenconcentrated~ by lrotary eva, ~.~ :a ... ;.i.;poration at 35°C to approx._ 3 ml and, chiromatographed on A small ;~,"rcoSuim~~n ('5~10~ g! silica ge~l) ,'. with ethyl acetate-methanol ('9~~9:1)! .~ , . ,, ,. . . . ~ : r . . .. ., ..- ;\Anialiquot of each 1i010 ml fraction was checked' for A-activity. added (the silylation reaction is very~rapid and does was then placed in a 1~ ml hypo~ vial and: 0'~ . 21 ml B'S~TPA~ with~ 1i $~ not r:equire~ additional heatin~g). ~~.~Anl.aliqwot olf~a,5~_w1 was~z then injected into, the GC-TEA. : The, conditions were: 6 ft x 1/4" _:V!j,~Jnterface T60°C, TEA pyrolyzer 5'20°C. ;;The carrier flow was 40, so:rb~~ W' HP,~ 8'~0~-~10~~01 mesh:;~ injection ~ port 17 01° C , o~ven~ 14 ©~° C-, TEA~ ;;L~ml Ar/min, the TEA cold trap was kept at ,12'5°C'. ~~ ,.. , x .~- . Tobacco Smoke Analysis radioactive fractions were pooled, evaporated redissolved in 2.0;~._ml acetonitrile. ~-,.An aliquot~ of' 0~.5~ ml .,, . .. . . (2 mm i.di. ) glass column packedi with,3$ OV-225 on Chromo- : . _. _ „ ...: , ~ ~ ~ ~~ ~~ .One hundred cigarettes or 40'cigars were smoked on an al MAin~stream~ Smoke smoker with~ rotating,~ he~adl,~ and~ the~~ smo~~ke~ was~ passed through two gas wash bottles containing 1.00 0 ml ethyl acetate and 1 g ascorbic acid each, one cold trap immersed' in dry ice and a Cambridge filter, ((69 mml). The ethyl acetate fractions LA ' ~ C ~.

were pooled, filtered and' evaporated to approximately 3' ml, ~. ~,y,.~ ;. and then chromatographed~,'sily]iated and analyzed by G'C'-TEA as , s-1-'jdescribed :yunder' "Tabacco' Analysis" ,.. - A ' s ~~~ ~ :}~ Sidestream Smoke F~:: ;- za b Cigarettes were smoked individually by `piston-type machine.` The sidestream smoke was lcd into 2 gas wash bottles at1 an air flow rate of 2'5 m1/'sec. late matter and nicotine in the mai.nstream smoke are the same ~ s~jr • ~„ : . .- Forty cigarettes were smoked in'a sidestream apparatus (~Thisair~~-~flosa through the~ sidestr~e~am, smoke apparatus with a chamber v~o~~lume~~ oIf 20I0I ml assures th~at y~ie~ldsof total Sf.r:,?: . i_. as those obtained from smoking of this cigarette in the open, r . ' air~),.°~ Th~e~ wo~~rk-ug~~ o~~f~ th~e~~ tr~apped'~, srnoke~~ wa~sidentical to th~at. 'of the mainstream smoke described above. Analysis of MHrDELA Preparations - 0 X xr . ~"The'commercial MH-DELA solutions contain 58% maleic hydra- zide diethanolamine salt and 42% "inert' ingredients" (;water ~ s~ etc. ); this~i equivalent to 3'7'$maleichydraaid'e. Five hundred mil acetone and NDELA-14C as internal standard were added to 1!0.1 gi MH-DELA solution and the mixture was subjected to rotary evaporation for removal of water. The concentrate was dissolv- ed in methanol andi chromiatographed on 501 gi silica gel with ethyl acetate as eluant. The fractions containing !3-activity were combined, concentrated Z~ 1..--, „)- to 3 ml and then again chromato- t1t 6 - ~' M.