Tobacco Documents Online

$ • • I e'- Q ," • o • . CGKFIII£NTI AL IlU~et eotalon on vhother It 18 sa,fo to add lu~lnO]L (gloves) to e~LSmrotte tobaaco Authu': De'. 7.J.C. Poe O~lUJ: Dad 0aY, obor, 1081 1o e • Is ,Umdmtlon 1.1 As • pot•ntis/ tabs• additive Bug•hal starts o2Z vSth the apparNt advantage tb&t It 18 • ~ursl produat.. Tk~s sdvsatsae 4. more sppsmt to • lqu/stm7 AuthorLt, than to • Tozlc~losist. The £sct thor olovos ny IN rood ~teoly Is aoak~8 and that flu•oriel" has bee• used wiclo17 by do•tints mi to • Iomr o1~oat bY do•toga (u n smtioeptic ud smtipFrette) prm~dej uguruoo that it is mot ILtah/y table whoa ads~nlstored orally. Also, allergy and ldlosynerstSo sensitivity bays so•nine17 not been prOblm. FOr b tOXlOolcq~Jt asked ~18 ouBenol alto as • tobacco additive?" or °Zs it ss~o to smoke el•yes?" the first sneers uust be (i) •sdoty via the ors1 route does not ensure 8s~oty via the label•ties rout• and (t1) what are the resu/ts at properly deal|ned tcud~ty tests? 1.2 Is this brief •p/hi•e. I trl to steer • course be•ran (L) • earns m gut-leslie4 that • 2or extra vhiZ£s o2 clove o~1 over sad above those fram • hot apple pie In not joins to dame4• nyoae'm health to a sessurablo extent sad (/1) an extremely ©sutious viev that a full rsmSs o~ safety evaluation tests SJ~Olv~q the Lnhalstioa route is needed before clow-s~oklng Is blessed by u ethical ca•party vulnerable to adverse publ/clty and lltisatlc~. 1.3 Is~ao-£aw u oua~nol hat bean uaod •s an smtimept~© and waak 1oca3 msl~O~tSe, tt to se:os•sury ~• my ~lov tO moss it £ram • toxi©OlOl~Cal vtev-point. (1.o. becmo It ks8 me biologir~/ activity) • 2. Tazleolo~©sl ovs~u•tio= o5 Eupnol 2.1 I s~osrapb by 0pdTks, O.L.J. (Fd. Cosset. Te~Leol., ~_1~. 54S-7, 1075) 13 • sensible s~urtin| point {J~nex 2). Yhe 5~portant ~a~ts ~n ~h~s noaoaraph are :- (1) gu|oaol vu Siva• sm ADZ o£ 0-5 ms/ks £or ~ood uao by d O

us)" {iii) (iv) ~CFA in 1~7. In I~ort terJ teedLq studies Is rodents, abe Xtvor Vii • tl~l~t Ol~ll |Or |i~ dOlN. A~O IDequll- atlam stud bamnOZThnSo o~ 8utrie op/tbol/~ van sees Leans wit& bFpo14Zo~ntosis o~ the ,mstnsaeh. Bug.sol Kava negative results £or sensitization the s~x~J~tsatiea test. But Koch et a..1 (Odon__.__tt. Revy., 31, 37g, 1071 and ides, tbi___dd, 34, XOg, I573) reported senAtivity to Bug.no1 An hemsmj. Bug,me1 obeyed roam cytotcmie ~tivity against lois eol2s. 3.2 Ac~crdJJ~ to Win annotation No. 3~77 in Yd. Cosset. T..ozieol. (28, 204, 2iff8) (Annex 2) based on & paper by Janiaud It 82,' the OTto-toxicity of Bug.sol Ls probably not depomdoot on opec/de £ormatioa and tn =st there£ore Indioatlva o£ possible e~clnoKeniclty. a.$ Kazan, O. (Oral Burg., 44, 7gg, 2977) reported that Eugenol can block nerve Lupulses, and thought thls might be due to permsnent nerve daneS., although elesrly I~L8 studies vare too 14~itod to estJdDlish than (Annex 3) R.4 2.6 Zuseno3 van re-eva2uated by J~C~A at its 1080Meetingand the ADI was • hanged to Temporary and reduced to 0-2.6 rig/ks pendtng the esteems st mKoLug ©arcinosenieity studies and ~urther motsbolian studies (Annex 4) JECF& reviewed Kugenol again st Its recent Z9112 Meeting. The results o5 a R-year oral ©arcinoKenl©ity study l• rats gore considered to bave provided adequate ovid.nee o5 non-carctnogenicity 5or the rat. One out o5 eve earcinopnicity studies (area rou~e) l• Lace 8eutequtvoc•X evidence o5 ponsible hep•tocarcinogen~eity. ThnnTeapors~TUsZatus of the ADZ vu removed but the lisle o5 0-2.6 as/MS left •s it yes (Annex 6). The nev data which led to thin decision in summsrisod in Annez 6. i 2.6 On behal~ o5 BAT, ZXBRA prepared a literature roviev on Euge•ol and 8s~role (Annez 7) and Diana Anderson emma.need on this l• a covering letter to Dr. ZveZyn (Annex 8). Whereas negative rewuZ~s ~or mute- ---~ C~ sent©lay gore obtained wlth Eugennl iteel~ o: vith oxeye oil in severs1 --- versions o~ the Ames' test, l• • bost-eediated assay'in sacs and in an Ames' test on the ~rlne o~ EugenoX-expoeed nice, • positive result van obtained in a test ~oz IMA repair in B. subtills. ~rJ ~J~

-3- In viov of this positive fl~diq end coo ~or elutosu~ctty in Alllm mLu• 3~dorsol roo~mondod sm t n vitro sane mutation .~ost in no•eolian oo118 sad sm in vt~ro test ~o~ el•steam•Jetty#sol•| on to in vtvo studies So~ c~astoKsnlclty i~ po•ltlvo results mrs shredded. 2.7 At ths roqoest o2 nyNZ£, an eminent expert i• mu&n~o~Lcity woo mmkod to roviev sll the data. Before so•lag •11 the available dmt-, this export took • poso4~4gtte viov about Uu/~noZ beeause of its s~ulZarity ~o Sstrsq~ole, u~lch had given • positive z~sult vith Drosophllla and bepstocollu3Ar caret•e-so in nice (Inn~ 0). and bee•use 88~role to vhlch Eupnol is arEuably •~silar chonlee~/y, ham sivel • eoutst~ntly positive result ~n • bsc~oz%•l poin~-smtsttau umsy. BocJ~so o~ dl£~ieulties In the 4~torp~ntstion of smts/oz~e4ty and careLuosenl~4ty data £or So,role md Emtrsjole, TJ~o ezpol~ suKKoJted that the best vsy to m~rt out the questions shout the activity o~ Rupnol ~tKht be to obtain, ~4rst, s ~u11 autagenleity profile of emtrs~o/e. ~.8 This rather tndlroe~ approach to assassins the aa~ety of ~ul~nol bad ~ittle instant appall tn-so-~s~r as sue couid onv~ss/e • considerable ozpondit~re o~ money and sti21 not be sure •bon~ Eugene1 itself. Yor~umatoly, however, the e~4nent expert revised has opinion in the 2tSht of the contents of Annez 8 and Annex 7. In • retread opinion he lis:s reasons £o~ bei~S no?s zolmd sho~t E~8s~oZ than es~rs~oZe and st£role. 118 advice nov Ls 1ors in harmony vitb that st Diana And~son. Re l~eOm~d8 :- (t) An Lu vitro assay - L5278Y or preferably hans• lynp~ocyte (il) And • mlcronu©leus easy In the ~oose Re m~psts Toxicol Laboratories (David Kl~kland) ne the best laboratory for those tests. X£ those tests are noKsttvo.sa he expects, then there would be no need at the present time to take the hatter ~u~ther. e Moo 1~ BAT ware proposing to put Zupnol In food, ~o~hs~snd£n~ the advice retorted to above, I ~c~ld have advised ~hat no further expenditure on tests is resXly needed 4. vlev o~ the 1~82 ~C~A decision px~vided ~hat anticipated de.lly exposure o~ huaa~s 4id not emceed ~Jme ADI of 0-2.5 mg/kg. R~mvsr BAT's intezest :elopes to tobacco and not ~ood. In this clrcmastancs meek 0 r%J

-4- Iraatett onyU~lnge~d• to be d~? 4o A buses saek~8 a cloys vt11 pre•anably inhale vspourland Eusuo2 plus some pyrolysis product• of gusonol that did not "got away' from the burning coal ~n t~me. Th• pyrolysis products vial probably be 8JJLtlsr tO those from s~y other sonstltuent• o£ sack• and I venial hardly tblnk it vorthst~Lle to undertake any special taxictty test• te ensure tb•t so ezooptlonslly tc=~e pyroZyai• products are £ormod. On the ot~er bud, dependLa8 on the concentraticm and totsZ dose, I an coaceraed that lok•led guseno2 may not be •a£e. ~ero are eve reasons 2or this. Ylr•tly, Zusenol jper so may be directly irritant to the respiratory tract, sad mocondly, thereus LaKmeted RupnoZ gee• tJ~rst to the ltver, abets it is uetoboltsed, tahsled KuSenol can reach the brain and other t$ ••us• dtr•ctly. 5o For these reasons, ] should not like to gee high concentrations or hlEh total doses Inhaled v~tbout st lout •one reassurance of likely safety from short t•rm tnhalsUon studies. The sort of study I vould think de•treble would be • 3 dose and control conventions] 90-day In, elation toxicity study in rats vith •peclal •trestles to respiratory tract htstopathologtcal parameters. 5• Havtn£ reached this point in By thtnkinK I was provided vtth ch-udcal saalysis data for smoke ~rms cigarettes coats~ta..lss| 7, 15 or 305 crushed cZoTes. These analyses confirm my expectation that cousidersble -mounts of 1•changed Eugsaol find their ray into the mainstream smoke, the 8mounts o~ EuK~nol in the smoke being proportional to the amount of crushed cloves in the tobacco blend. 7o ]~ron the data provided one can calculate that s SO Eg person 8~ok:Lug 50 cigarettes made of tobacco containing 3~ crushed cloves ~ould inhale each day: 50 : 4.05 mK Eugene1 .~er day Q or 4,05 eG/KK body m|ight per day Th~s wouAd onAy slight2y exceed the ADZ for orally •dmtn2stared Eugene1 set by the :1982 4"£C~A Cammtttee (1.e. 2.5 me/ME). m~b =smudD ~J

Q So ] have sine been shown data for the mutslenictty of condeneatea derived from ci|arettea coats/sin6 varyJ~E levels of cloves. Z have no stoat faith In the tnterprotahilit7 of these data u far am nan Is concerned. However, I note that the mutaaenlctty of the comdonsatos variee invlrlely with the level of incorporation of cloves in the tobacco and that this pattoz~ has been consistent in repeated stud/as. I reK~rd this findin| ms reassu:inK and encoura|tn| but not s JobstitBt@ for more cemventLcmal tox£colosioal studies. D. Finally. I tmderatamd that sooke Inhnlatloa studies sure in pl~qLt~sa. althoup I do not know the details. These seem to be conins up wLth the result that the smoke from clKarettes containlnK 165 Eusenol and fl-am control ct|arettes do not 8~esingly differ in their apparent affects on, presumably, rats. )0. Before gtvint n final opinion on Eugenol I feel I should Zi.ke to know ears about its metabolism after ingestlon and after inhalation. I understand that after oral administration it can be demethTlated in the liver and that most of it is excreted as ethereal glucurontdes in the urine. Is the same pattern evtden~ tiler inhalation exposure? II. The fact that Eu~enol is a phenol does not prompt me to be concerned that it aipht act as s tunour-promoter since such activit7 in relation to souse skin is ltadted to phenol itself and to a.fev other quite simple members of the phenolic class that uuse marked opidemal h.vperplasia. In the 1950's I applied clove oll to mouse akin and was unimpressed by its ability to cause epidermal hyperplasia. However, I seem to remember that In hir~ dosaKe it could cause death through lung sedans (H.__BB. I do not have immediate access to the data and 1~ say not have been clove oil which caused lung deaths) 12. Provisional opinion ! There does not seen to be too much to warty about in l~lat.Lon to the inclusion __. of crushed cloves i~ cissratte tobacco. However, I believe tha~ the relieving steps vould be advisable: 0 (1) Cempare metabollmn after inhalation and oral exposure. (.e4 ill, limPA__ J ....... A |~. AO'111 ~ji~ BJOAi;A~I Ilii~

(it) ¢lS~) (Iv) (v) -8- CampXoto 90 tnbsXstton toLtctt7 j~udy hip ~tJl~l the limits Nt 1~7 ~C~A'I ~ O| O - J.| It|/l~| CaZT7 out tn ~lt=ro mutalenicLty and e2ag~oleniotty testa tm nemmslia: cells. Check exposed human populattOaJ £cn" evidence o.• tZ]keriK- enicity. 8tKned: Dire: rrawnij 3.C. Roe Din, DBa, 1~C. Pstk. 2~d OaZobeT, 1983

oRc^ lSA'rlO MONDt^LE L^ S^ TE l JOIHT FAOtI~HO EXPERT CC]~L~ITTEE ON FOOD ADDITIVES "t Iome~ 19 - 26 April 1982 EUGENOL txpls,utiCm This subst•nce ms evaluated for •cceptable daily intake for man by the Joint riO/IWOExpert Oos~ttee on loud Additives in 1967. •rid 1979 (see Annex l, P.ef. lh and $1), & toxieolo$1c•l mono|raph uss issued £n 1980 (see Annex 1, Bar. 52). Additional date have become •v•ilable and •re suuwtrined •nd discussed in the folllotrlng monosr•ph. The previous aonosr•ph his been expanded and is reproduced in its entirety belay. ~IOCREKICAL AS~CTS Abscnrbttonjuetsbolisu and distribution lntrap~ritoneal injection of • sin$1e 650 us/ks dose of 14Cuethoxy labelled engenol resulted in rapid distribution to 811 organs. Both •shot and rater soluble mattrials nero recovered froauost tissues and excretions. Only 0.2-l.0~ of the dose vie ellu~usted as ezpired 14C02 (~einberSot el., 1972). Over 70Z of a lethal dose ot tusencl ms recovered on death, frou the urine of rabbits (Schroder & Vollner, 1932). Adulnistretion of single 200 mS doses to rats led to increased ur~naz-y outl~ of etheTosl |lucuronides of 33-35 ~&/rat in 12 hours compared to a control value of A aS/rat. Ester glucuron£de values uere unchansed (Yules, 1974). Studies carried out in liver uicrosoua! preparations froumale and feuale Yischer rats end CD-I =ice shoved that foz~ation of eu~e~ol 2',3-cpoxide frou eugenol occurred Ln just detectable snounts (Svanson et a~., 198D. Epoxldlz•tlou o£ sultan] by rat l~ver cell cultures has been reported. The dihydiol mctabolitc of eusonol has been isolated frou liver houogenstes sn~ urine of rats prctrea~sd tr~th eugensl. These metaboI~tos ms7 arise fro= the action of epozida hydrase o~ the euleno~ epox~dc (Delaforse eC el., 1980). Incubation of eultnol ~r~th rat l~ver epithelial cells resulted L: p:oduct~on of 4-(2'-3'-dihydroxy)propTl-l-me~hoxyphenol (Jan~aud~ 1~1. : ".. ~,~'t st Ibis docunmenl doq~ nol constitute • .,,n..~ pubhcath~n. It shouir, not be rev6ewed. ..: ~:,.;te~J Oe Q~O1e(J wQIho'.J~ ~the ~lmlr~M O| • .:~ni~LAI lot VW~W expressed in signs~ Ce document no ¢oml~tua pas ~ pub|~.atio~. IIne dolt lane I'o~et d'aucun compt8 ;endu ou alumni ni d'aucuna citation sans I'amtoriution de I'Orsanisat~on mond;ale de la Sanlil. Les opksiom exprimees dam les articles signds n*itnSag~nt Qua ieurs a~tevts. mmm mm~ (..* C: ....... ~ Jr---- I fti • ~ |l------i,~ dlStSi~

JIOCAE~CAL Z~CTS The pharucotosic effects of eusenol i~tude the prevlous1"y revised inhibition o~ B-D-Ilucoa[duronic acid conjuration "Ln ra=s receLvinS 150 m~aniu~t (Hez~cixla et aZ., 1966). Itydroxylatin6 activity of liver homosenste on dius~hyla~nopyrLne or hezobxrbltal use depressed in tissue from ulce dosed rich 160 m~kS of eusnol and sacrificed after one hour (Jaffa st el., 1968). Eugene1 had no effect on 8minopyrLue-lt-deumthylatton activity In the liver of rats Si~an about 1OZ of the LI~0 three tiJuts daily for 2-2/3 days. There use s alight decrease in hexobarblt81 1stere! deflection time end in urinary ascorbLc acid content. (Gruebner, 1972). Y.uSeonl uss reported to inhlbit respiration in vitro in sdtochondri8 isolation from the liver o£ adult uutle, Charles k'~e-~'~s. CoBcantroclous of from 0.11 to 3.50 .nil o~ eujenol vert present in the suspension 88diulBj inhibition of respiration began at concentrations of 0.88 nil (Cocmore et al., 1979). At 1 ~q concentration, eugenoI ums reported to CRUSe a 61Z inhibition o[ norsdrens~£ne induced oxldstivometabolism In isolated bream fat cells from adult hamsters (Paterson et el., 1980). lntraperitone81 injection o! 200 mS/kS tuSe~ol i~luced anesthesia in male S~ss albino mice; the mean sleepin~ time in • group of 10 dosed an~mals we 17 u~n. Tvo o~ ~he an~ssls died vlchin 24 hours of treatment (Sell ~ Csr~in[, 1976). Xncraperltontal admlnistratlouo~ tusenot is also associated ~th hypothermis in race 8nduyorelaztionand anticonvulsant effects Ln mice (DallmeLer & C~rlinl, 1981). F~senol is used as a dental analieslc (Tyler et el., 1977);. the toe,pound relieves pa~n from irritated or d~seased tooth pulps-but £s not a t~ue lo¢al a~esthetLc (Sticht & Smith, lgYl). Subcutaneous injection of ~0 mS o~ eugenol di[27 foc 7 days (total dose 1365 ~slks body veishc to partiatly hepatecco~ized me~e, Charles ~iver rats had'no effect on rats of liver regeneration (Gershbein, 1977). Subcutaneous injection of 0.t ml of p~rified eugene1 £n adult Walter Reed ~h~ts rats caused necrosis and inflmcion at the injection site (~ebb & gusselj 1981). re.m*

• .-,,Backsround paper 27 -3- Other effects include: Animol Dose 1touts Zffect 1of stance Vice Rat Fros ca. 50 ~/kS t.v. 10o-3~o =s/ks i.p. " "5o -S/kS I.p. IOO =s/ks i.p. 16o =s/kz t.p. 200 us/kS i.p. 0.1-100Z direct nxposurt of nerve CholeresLs leductin in rectal temperature l~raaoe in sleepinK pentobarbltal- 131Z ethanol- 120Z No effects on spontaneous motor activity Severe depression and paralysis of hind quarters Catatonia Blockase of transmission of evoked impulses Ln exposed sciatic nerve Chabrol. 1931 Caujolle 6 MeynLer, 1960 Sots. 1969 de.qello el'el., 1973 Kossm, : 977 TOXZCOLOGICAL STUDIES Special studles on uuta|enicit7 Zujenol yes nesa~ivc in a Samonells assay employing four mutant 8tralns (T&-lS30, TA-1$31, T~-1J32, T~-196h) both directly and after the use of mouse liver postucLtochondri81 fraction for'activation. Zt v88 also inactive iu 8 host-medlated assay (Crete & SaYS|to 1978), Eusenol yes also reported not to bomutsgsnlc in Salmonella T&-100 in a liquid suspension assay vlth or irlthout an S-9 fraction from Aroclor induced rat liver (Edcr ec el., 1980). Negative results wrc also reported ~th eugenol using Salmonella strains T~k-98, TA-1535 and T&-100 in the m~b

|~ckgtuund paper 27 em~ o plate assay system vith or vLthout liver activation frou Aroclor or 3-methyl cholanthrene induced tats. The 2',3*-epoxide of eusenol yes mota|enic for etraLn T~L-I$3S Ln the ab•ence of 8 liver activation system (Sv•nson ec el., 1979). tuseno! yes reported mot to be uuteSanic to Salmonella •trains 1535, 1537 or 1538 vIth or vlthout • tit liver act/racism system. The 2',3'-epoxyderlvetive of eUltnOl yes mute|celt to str•in T&-l$3$ vLth or vJthout lives 8¢tivet~on (Del•[orse, 1977). Specie1 studies on cerctno|ntott7 Groups of about 30 youn| adult female CD-1 mice mere fed 0 or 0.SZ • ueenol In the dlet for 12 months. Other Stoups recelv•d 0.0$Z phanobarbitol or O.SZ dletaz7 euSenol £noddltion to 0.0$X phenoberbitol In the drlnklu8 rater. The anJ~81e received control diets for an additional 8ixuonths follovinS edu~ni•tr•Cicm of the teat diets. In the Stoups s/yen only phenob•rbitol, 3129 nice developed liver tumors, uhilt no liver tumors vere found in the other 3 Stoups. The liver yam the only or|in examined for the occurrence of tumors. A second experimnc yes conducted in v hath groups of 40~to 60 male end 40"to 60 female CD-I ulce rare siren 0 or 2.S micromolee of eusenol tv£ce vukly by Striae 8tsrtinZ at 4 days of ass and continu[n| until 35 days of ale. The 8nJ~utls vere theo maintalned u~thout dosing until the experimmnt yes terminated at 14 months. No effect of trestMnt upon the incldenct of liver tumors vns noted in either •ex. 2n a third study, Broupe of 40 to $0 male CD-I mice vere injected i.p. at 1, 8j 15 end 22 days of eke vlth 0.63, 1.26, 2.52 and 5.04 micromolee, respectively, of eusenol or eugene1 2's3'-epoxide. As compared to concurrent controls receivinj the trioctanoin solvent only, neither treatment eroup had•n increased incidence of liver tumors (Hiller et el., 1979). The ability of euzenol to promote skin tumors v,s studied using |roupe of 20 fetlee ICK/HA Sviss. One Stoup yes liven einsle cutaneous initiatln~ dose of 7,12-dimothylbenz(•)anthrecene'(Dt~JL) to the back. This group and another group not /else•ted Irltb DM~ received 3 times weekly cutaneous applications of $ mZ of eueenol for 63 reeks. Ho carcinomas vere fou~ in either |roup and no papillomaJwere found in the animle receiving onlF euzenol. Three animals developed pap£11omas in the group inltLeted v~th DH~A and al•o treated ~r~th eusenol. T~o papillmws end one carcinoma developed in control animals initiated rich D~ and then treated three times veekly v~th D~SO, the solvent torero2 (Van Duuren et el., 1966). In another study carried out in the same laborato~j, eusenol ~as reported to have a l~rtial inhibitory action on the csrc£nogen~city of benzo(s)pyrene vhen the compounds vere applied to~ether in a carclnozen~c skln paintin8 study (Ven Duuren & Goldschmldt, 1976). In • limited study in m~ceo eu|enol d~d not potent[aCe the tumorigenic effects o! methylcholanchrene (H£tchcock, 19~2). o~ r~