Tobacco Documents Online

4 a / 1 T= Cozacu. FOR TosAcco RLstARcu-T.S. A.. I.c. 110 F_tST S9TU STRF.F.'T \"Lh' TORK. .V. T. 3002: Tebruar,, 25, 3r°"3 Freddy Hoobu_yer, M.D. President and Di ector Bio-?.esearch Cor:sulta:.ts, Inc. 9 Co=ercial 11venue Ca=bridge, Massa:`.;:setts 02141 Dear Fred::y: I L:.ders:a.nc yo_ have been ac:-ised we have :ae- fu_:.ished cepies of yo•_ letter to N_. Ecxin . Caccc da:ed Fe:,:,.Lry , 1373 _.., of you= b:ociure entitle_"I:.halatcon Studies wit.i Cicarette S:a'<e - A TycF_ical 8ulletin on Haso.•s Used =y 3io-:esee_ch Corsu:ta:•.ts. Inc." Also L aZ infor.,ad L`lat Dr. Gard.ier in his conversation wit1 vou ir. Fe•- Haven on ~i,ra! 22, 1973, discssed wi_- you the r.ecessity of co-c'_e:`_nc t'r.e wor< called _-,ce: o'_ cont--ect =.._ Determina:io: of the Use:•_l.r.ess cf the Gol_e:: Syrian Mirs-az as Model An.L':al :-r I_hala_ion St•:Zes" =e=ore a-:v dcanit_oe con=2usicrs •zm be sade. 5uch cenc'_'3iaa &re :+ecessa-: be?ore L^._: .____sio- can be rsde as to ex: e:?- hzs!:s. __n__:ion =e'_L=ing to cica_et:e s-t'te ex^csu_e of -.n vou_ '_e_:e: to Y.:. ,:__tb and in yo.- :'ec~c-_ =--1e=_.. ;ou t'_ of ol._ c_ ____t re!_- - as -0_1_ _. "nL_ Cnt2 Ca'lelC-:.e.-.S. or ...._:: e_ oz r.ot v•:er._=- - -y --o-=.ese-c-. .,_, _,- ur.=ec a-'e=-.•' _cisa o`, o' as a res_'_ w'` .. =e .-e sc_e ancaxclLs::e oe: .,_ _-e Cc_zc-- =-_ _c5acce Resea==i - U.S.-.. B:c-R!sea:c; Co.'.sL'Iti-.:a, In:. _-=ees to ":ee? sec::_ L:._ cc..=-_-n=ia: nny __c:,s:'_o-, k:.o;lecse or :___ o::---rY in eon.._:_=n ~i_.i e- as a res•_lt c'_, .ork done un'_e= t:_~s fu=ther __-__ r:c= to use suci in:or-.ier. 'er _tse:f or ~ c_.e=s a:t.. _`'=ect_': __ __ to a:r• ..__c ;ar=l c:_-_e c: .^: :oun..__ :c_ cisc_c•e ----c== Reses.c = - C.:.... wt _.oLt The :o= w=- --r. ":.11 e:uicc.e:.t dce--jents, books , :eccrds , notes or ot-he: aateriais or dats furnished t'y or on be:zaif cf r'e Cointil :er Tobacc= Res!nrcn - U.S..,.. in r_n necticn with work eme _..._- this e?see=ent s^tl: at all tices be the c^=e:t': of The CcL:.nci'_ for Tcbscco Reses==: -.._.?.. a_nc siall be za:'_nec -3io-=esea:c:. Cc-.s'::=a:.`.s, Inc~ to -& res:=_==-~e ovr.ez as desicne:ed by The Co'ncil for Tobaao ?~_esearc'.^. - C.5 A. at _.e ter=inatio.l Cf t~hls L~r3i:..e:1t or at t.: t:2 .__t ne CC_.___ for R.e.seare'i - L'.S.a. mav _ecuest. CTR CaNTRRCTS 0057416 11225457 W W CTR HN 042-407

a /1 . / Tor CoLxcn. FoR Ton.tcco RESC.{rcn -U.S.A.. I.c. Freddy =cdc._ger, M.C. - 2- Febr:a.ry 26, 1913 Consezt has not been given by us to yo._ disclcsure of information. ciovledge or data obttir.ed in connecticn with or as a result o_ work dor.e under our agreement nor has consent been given to use such information. As you knoc+, the work to be performed under the contract was in the nature of a feasi*ility study. To date, I am informed, the results are incooplete and inconclusive. Your Technical Bulletin refers to t~rie S4+altoa:lorrissey saoke exposure machine ycu have been using and states among other things (page 2) that this is your machLne. The machines you have been using are our property and are subject to the grovis:ons of the second paragraph quoted above frcz our contract. As you kaa-r, we also own the oatent on the machine. The eaczines used by ycu are not the kind to be used ic future work, there having bean surstantial i= rovements since the aode: tsed bv vou was const^:cted. C,Lz relations vit`h you and your co7-pa.nies `.:rocch the yea_s have been cor:_ial a:._ we cislike having tc write yo_ i- t:.e vei=- of t`.it let.__. However, we a:st in:ist t-hat ar.y partv enter:n; :rn a c•ontract rit~ us ad:^e:e to the te=3 of that catract. Sincerelv. i1 . . . E:",.I._ =xe=:ave t•ice .:es_'Ze-t "^=: • _•. CTR CONTRACTS 0057 i ~ 11225458 M 9R I-IN 042408

V ; LNF-iAL.ATIGN STUDIES tiVITH CIGA ;ETTE SMO:CE 7 ~ A Techr.:cal B::11e::_ ~ ~ 3 o.^. ^.1G:h:ds L'se:! t.;' i : ` ~ ~ 7 ~ B:..-Resea: cn Coas_lt~ :s L-c. t , 9 Cor.:ne: cial Ave_ce i Car.:b=icge, Mass. CZi :'. E (617) 864-8735 i ~ ~ i i I CTR CCNTRRCT6 005689 11225430 C TR HN 042409

INTRODUC=?O\ Since Wynder's studies on mouse skin tobacco carcinogenesis (1953) the application of cigarette smoke condensate to mouse skin has served as the only relevant bioassay of tobacco smoke carcinogenesis. This as well as'related test methods have been reviewed repeatedly; tech- niques have been standardized and compar able results have been pub- lished from various laboratories. However, those ernploying mouse skin painting tec :.nology for carcinogen assay of cigarette smoke have always been aware of the gap which exists between mouse skin and respiratory e?ithelium,and efforts have continued to develop inhalation techniques which could replace mouse skin pair.-_4=g and cause cancer in the respiratory epithelium : o: srzoke-exposed ani- mals. Such i nhalation technicues in add:tion would serve to study ac_te toxic effects of cigarette srnoke and its poss:ble role in the causatio= of various respiratory ills other than cancer. These inhalation tec~hr.:c•_es are now available, procedures have been standardized t-.d neoplastic lesions of t:.e respirato-y epit`:eliurrm ha•:e been produced vit : fr esh cigar ette smoke in at least two separate l::o- ratcrics. Dontezw:l'. has reported the induction of epi:hel:a: c=anges in the 1ar~:~~c of ra..do-bze= S}•-ian r.a_:.ster- i-%c1:._.=y early -..:asive carci:o:::a (_.=C Sysr -~esi::c:: Series No. 1E, A_:-~-I 1S70i. _..- sa-ae asthcr reportec -_...- lar observations in large- ber s of =:-:s:er s at a cor!erer.ce at :--, 5etl:esda, i;eve-.'oer 19'2, .:-[t:: a: .nc:dence of 1U. E;o o: invas:•.•e cincer of the laryrx in ::a:ysters eapose: to cigarette s:noke in the Fia--nbur g type Ii s-noking -nac=ine. Bic-Resear ch Consultants, Inc. rece_t'_y co nfir:ne: Donten.vil:'s c:s_c vation s us:--.g ir.br ec Syr ia- hamsters of two car cir.ogen-suscectib'.e s:rair.s ex-nosed to smo::e from Ker.t_cky re:erence cigarettes -... 101 weeks. 1'~hfle our trae incidence e: invasive carcino:sas o: the :a: rr_Y is not as yet known since the experiment is incomplete, we have obs er ved undoubted instances of pre-malignant char.ges ir. the lary=r. We als= observed large rr.aligr.a-: tumors of the r.ascphary _e never previousl•; noted in untreated har:•uters of comparable age. It is thus established that, contrary to expectations, passive iahrlaticn of cigaret!e smoke by Syrian ha-:zstera is a useful bioassay system for tobacco srnoke carcinogenesis. This is an importaat development i:- - 1- CTR CCNTRRCT9 005690 11225431 . CTR MN 042-410

a /r, those interested in co.^..parative evaluation of carcinogenicity of inhaled smoke. l~ot only are results obtained in respiratory epithelium more directly relevant to hu.-nan smoking experience, but through the use of native cigarette smoke nuay possible artefacts of condeasate prepara- tion are avoided. In the course of chronic smoke exposure studies on Syrian ha.-asters, it is also possible to study physiological parameters such as lung function, breath sounds and electrocardiograms and thus to investigate effects of cigarette smoke related to cardiovascular and pulmonary diseases. Bio-Research Consultants has unique experience and laow-how with chronic and acute i=:a:ation studies using its own Walton-viorrissey reverse smoker to si-iulate human smold.zg and to yield knowt concen- trations of fresh sr-..o;ce. Well defined inbred SyriaL 'naraster s are used to assure reorocucible results. The'cost of such itihalation studies is surprisingly low consideri:.g the cornplexity of the technology and the large zur:be.-s cf a=,-:sa.ls r;'hic: are necessary for statis: callly signif:cant results. The use of _:::oke .: ^.alatioa st•:d:es is recor.= ended to c:sarette an d cigar r..z=.u?acturer s as weL as to filter ma.-.;:fac~.:r ers for the bioloi:cai defi-...,.. e: the fresh smc;ce generatec or by their product by acute a.-id!or chro-•'; bioassay for bra-d compariso=, eva_•_at:on cf :-c-?:- ficati..ns of cigare•.-:es z-._!or f:lters an_ :or c•_alit;• ccntro'_. bioassay, p-oper1;• do=e and ir.ter~-etec, reveals more aa=rcduct t: an F_r., c:e-nical or ,:ysica: ::ieasur eme=ts. 1) C:-_:ce of the e~,.~eri--:er.tal za:nal It is ir^per ative t::a: in!:z;atson s:u=ies are carried cut in s:f':cientl;: large populations, rather than in sma11 =u-zbers of exper i:ne=:a1 in order to permit prover statistical evaluation of the results obttined. Since the costs of such studies v:th pr ^ates, dogs, cats and ot_er le-ge- sized animals would be prohibitive, s>•::all laboratory a.^?-na1:, such as rodents, are most suitable for this purpose. Among the rodents, the Golden Syrian hamster (Mesocricetus a::ratus) assumes a special role .wnich makes this species pa.-tc:::arl;: - Z- 1 7 CTR CaNTRACTS 005691 11225432 CTR ~~~ 042-411

A 1 for research with tobacco smoke: the hamster is from ten to 23 times more resistant to the toxicity of nicotine, when injected intraperitoneally, than the mouse, and from 1. 6 to 3. 6 times more resistant than the rat. The differences in nicotine toxicity between these three species are in the same direction, but even more pronounced, when nicotine is adrrin- istered to the animals by inhalation of whole ciearette•smoke. Conse= quently, hamsters can be made to inhale substantially larger amouats of smoke than mice or rats. The advantages of the hamster for smoke inhalation studies over other rodents are further enhanced by the fact that spontaneous disorders in general, as well as of the respiratory system in particular, such as lung tumors, broncbitis, pneurnonitis, etc., are virtsally nonexistent im the hamster, but occur quite frequently in other rodents. It is generally recognized today that the use of inbred strains of esper i- mental animals in toa-icology yields res-alts which are rnnch more readily reproducible than those obtained wit:: r-oninbred animale. Furtherr.:cr e, the susceptibility to certain responses of critical ir-..portar.ce, e- g. •.=e causatio:: of laryngeal tumors by cigar ette smoke, ard the resistance to undesir able side effects, such as nicot'=-e to:cicity, can be significz=tly incr eased by a proper choice of the :: ost sui_able :nbr ed strai:n or s:r zin-. Hence, iz'r•a-nsters a higher dose of s-zoke can be saiely adrr:nistered than irn c='=er rodents, and a greater respo nse will result, both becavse of the higher dose and the increased susceptibility w=en appro?r:ate i_- bred strains are used. The ava:labil?ty of inbred strains of : amsters arid c--- fa.: ±lia•ritv ab the prore-ties of these i..'--rec lines are of great adva ntzge, t::er efcr e, tc t2a e most efficie.^•t and ;ro'.`.ious conduct of sT0<e L'L1aLatiOn stud:es. «•o of t'r.e inbreZ strains of hamsters st•.:died by us, i. e. the 15. 16 a^•= b~. 2C lines, a>?ear to be particularly suitable in this respect. Unless there exist special reasons to the contrary, the studies v-:'.1 i=- volve r..ale hamsters only. Fer-rale ha-mster s may be included or :na': replace the males altogether in these izvestigations. The use of :e:-ale hamsters would increase, however, the costs of the study, because fe- males, ir, contrast to the situation in r-L{ce ar•d rats, muse be housed in individual cages to prevent fighting, «:.ereas males r•.:ay be kept in lots of six per cage. -3- cTR CoNTRacTS 005692 11225433 CTR 1-IN 0424 12

I 41 2) The smoking machine We reco:runend, but do not limit ourselves to, the use of the Walton- Morrissey reverse smoking machine. In fact, any mechanical device is suitable which vrill allow exposure of the heads of the animals to exactly known concentrations of smoke (up to 19°.'a of t}:at concentration of smoke which egresses at the mouth end of the cigarette during each puff), and which permits periodic alternation of smoke and fresh air in the exposure c'r.a:nbers in exactly adjustable and reproducible fashion. The Walton-hiorrissey reverse smoking machine operates on the prin- ciple of pushin¢ air through the lighted cigarettes, rather than by dra.v- ing the air t:zroc:¢i: them, t::ereby considerably simplifying the overall mechanical setup o° the smoking z-.:ach:ne and avoiding passage cf the smoke through valves. r Most of our chronic ir?:alatio_ studies were perfortr:ea with the cigarettes s:noked in a vertical positior.. Recently, the machine has bee n L:apte'_ to the use of cigarettes smoked in a horizontal position, v.-itbou: modify- ing the main chara=:eristics of t:e machize or of t=e exposur e c'=tmber. 3) The exaos;:r e charnbe- The e!c:es::-e c.`.a-zber for ha-s:ers c: the s-:c?cer accorr~oriates six a_.-:a:s s:...;:'.ta_neo si} lt is less ..a.. two .__..es f:or.m the r^^•.:thpiece of the c:garettes, has a vc_ .zrne of 72S r..l ac: is fitted K•ith a s-all raagne:ic The ha-ris:ers : a••e only t_eir heac.s inserted into the e~osure char..•cer. is achieved by fitti::c e!c:. a-i- r.:al per:naner.:lya fe'-t-alu-zir._:ri colla:. __fs co'.la- ca=n be ia- serted into a.•o1:e-liice openicg '_n the chamber, vr'ae: eb;: t:•:e a:;-:.als a: e retained wit: t::ei- heads ins:de the exposure c^a-sber :cr -ae d•:-aticn of the exoos::: e. These collars have to be reolaced fro^^ tirr.e to ti=,e, as the ha.^..ste: s increase in size. They do zot iate:fere •~iti the well-being of the ani- mals, and hamsters have fr eouently beel knou:i to r..ate and r eproduce with the felt-alun:i^u- rings in place. 4) Cond:tions of smoke exoosu: e Smoke is generated In 60-second puff cycles. Durir•g t'ne t'•%'o sec- or.ds, 3: ml of air are pas<_ed t..re:-yh each lic :te2 cigare:te: :~~nen four cigarettes are smoked s_i-nulta=eously, a totai of 4 x 3: - 140 .:1 0: air r r cTR cOh(TRRCTS 005693 ° 11225434 CTR I-IN 042241~'~ ,

a /1 pass through these four cigarettes, and 140 ml of smoke are emitted into the exposure chamber of 725 r:a volume, thus resulting in a 140:725 or 1:5. 2 dilution of smoke in the exposure chamber (smoke concentration of 19.3°,'0). The smoke is maintained in the exposure chamber for an additional 15 seconds (actual exposure time) and is then replaced by fresh laboratory air; this exchange takes from one to two seconds, and fresh air is continued to be circulated through the chamber for a total of from 30 to 33 seconds. Fi.ally, the animals are allowed to breathe smoke-free, noncirculating air until the begin- ning of the next'smoking cycle, 60 seconds after the start of the previ- ous one. Six hamsters are being exposed simulta.r-eously in each exposure char-..- ber; the same six ani.:.als are also housed in the same cage. - By tic=g into account the known average values for r espir ation rate and tida! volume for adult harnsters, it c= be calculated that approximately 104 ml of the smoke-air mixture from the 725 ml eocposu:e chamber, or 14.4170, are inhaled and ex:.alsd by all six hamsters duri-g the 17-seccnd period of smoke generation and smoke exposure. 'r'ence, there is only very little chawce of smoke being r eincaled by the ani-zals. Assu:z:i: g a carbon -or.ox}de coccera: aticn cc tppro.,e_rr:ately 4°,'e c 1'a !v/~ l in undi:_:ed cigaret-:e s:^c<e, the CO concentration ia the exposur e cham- ber (::5. 2 dilut:o=) a--o;=.ts to C. 7 7070, or 7700 ,-.- (parts Der r:?__.-=`. Let.`.a: carbon r:e-exice concentrations (see i?a=.dbook ior Enviro =-:er.tal Biology) a: e!istec as 6000 00 .. :m dogs (in 20 tc 3C minutes), 4000 pp.m in mice ( in 45 ta 50 r.:-nu:es), and :"v=-:~ pp-:z -= ra:s (in 30 mir.::teai It is obvious tha: t=.e cLrbon rnen~xide :oncent: ation under our ex=cs~r e conditions of the hamster are rather close to t:e le::-.al concer.trat:o::s, and that carbon monoxide is indeed t*-e lir.:iti.r.g °actar ..n smoke tion stsd:es in the ha:r.ster. This is confirmed by the fo11o•.c:r.g two indicat:ons: (i) all ar.i--aals :.ave invariably and instantaneously died in t::e rare event that a mechz.:cal failure ir. the s:r.oke-re:_•soving cevice a: t~-e end of t=e 17-second pe:icd of smoke generation ar.d smoke exposure caused the animals to i,: ale smoke for 60 seconds or longer, without allo:ying them to breathe f:esh air; (ii) even under r.or:nal opera:ing conditio: s, v.•: en the animals were allowed to breathe fresh air for 43 seconds out o: each ont-mir:.:•:e :u:: cycle, blood carboti~.•he-ioglobin : ose tc re'-ati•: e cencer.trations o: fro:•:z 20 to 401,'e after 20 smoking cycles, but decreased to about 5°,'e within four hours and return ed to normal (below 1r.) within 2= hours. - 5- CTR CaNTRRCTS 00S694 11225435 CTR HN 042414

a A1 Based on these considerations of carbon monoxide toxicity, as well as on our experience with the mortality and the body weight changes ' during chronic exposure of hamsters to whole smoke from Kentuckv 1R1 reference cigarettes, the most appropriate schedule of esposur e to smoke from this type of cigarette in hamsters is as follows: i 1 I Each•anirr:al is exposed twice a day (with at least four-hour intervals), five days a week, to eight consecutive smoking cycles of one minute, consisting pf two seconds smoke generation, 15 seconds actual smoke exposure ar.d 43 seconds exposure to fresh air, involving eight consecn- tive pu_°fs (second through ninth puffs) of 1R1 Kentucky reference ciga- rettes, at as acturl smoke concentration of 19.37% of that emerging di- rectly from the mouih end of the cigarettes. A schedule involving exposure of the ani^ials on seven days eacr. week is feasible, but adds considerably to the costs e: t:e study. In most of our previous studies, srnoke exposure F•as starte'_ w:e= the ani--ia1s (males) had reac.~.eci a body weight of 107 c 3=, i. e. G•hen they were near or practicaliy at :••eir adult weight, and when t?:e}• acere about three mont`s o° age. S:ao:ie exposure was coati^.ued for periods o: from 72 to 92 u•eeks. Ther e exists no reason c•hy e--oosur e co•-Id aot be startea at an earlier ace, except t: at the i.:tial rnortalit}• wo•,:1? _:cb- ably be : igher .•• this cas e. The morta?ity of t: _ anL:•a:s -was very low in the bes-sr:n.g c: the it za::¢ed f: c- 2.., 7r', a?:er -. %s•eeks o: exoosur e a== from z to 1_ . z_her 60 weeks, but s:art-_ to increase considerably a::er bJ .c•ee::s of eaposure (31 to i2'e). In orcer to assure a su_icie:S r•:::aber c: vcr s after 90 weeks of e»os:re z=d to allo•x• :or a.-.= a?s to `oe sacr: :iced :or histe?e.:ho:ogic s-_'_?es at intern:edia:e time intervals, e. c. a.aer 45, 60 a nd 75 weeks c° exposur e, it is necessa_y to star: ti e e.- per i^:ent with z>?-oxi -ia:e1: 1CO a ni-ials (since ex-_~osur e tc s^o*-:e '.s carried out in lots of s•v: animals, t=e ir.itial number of ar•imais .:c be used sl:ould best be or•e divisible by six, i. e. 102). 5) Gas ohases of s-no)ce The 1'lalton-I`iorrisse}• reverse s:r.o:<ing machine le_ds itse!' to e.?osur e of anizr.als to eitner .~: ole c'-garette sraoke or to i:s gas Dhase, i. e. to the portion of s:noke re:r.ain:n; after passage o° smoke thrcr--h Ca.:.- bridee or analogous filters. - 6- CTR CCNTRACTS 005695 Z W 11225436 C TR HN 04224 1 S

! 6) The use of ci¢arette filters Cigarettes may be used with or without their own filter in place. 7) The use of ciaar smoke Adaptors for the use of cigars of all sizes may be rnanafactur ed and exposure of ha:risters to cigar smoke, thus, becomes possible. 8) Control Qroups Two simultaneous control grc•ips appear indicated for each type of ex- perirner.tal ani:aal used, i. e. for each inbred st: ain and sex. The fir st control group consists of an equal r.umber of ha^ister s(102) of the same strain a-.d sex which are treated in an id-tical fashion to the exper imental ani-mals, except that no cigarettes will be inserted in the smoking r.,ac: ines (s :arn-smokec control a~:mals). The anin:.a-lis of the seco=d control group receive no treat::•se=t other than that they •x^:11 wear t:e alu^inum-felt collars (cage-held control a----..als). These ha:nster s%-i11 also be c' tie same strain and sex as those c° the experimen:al group. F=e•.zous studies have shown that the r-..orta'.::-: o: the cage-heid contrc: a_i-•als is r.o lower than tha: of the ar.~-nals c: t:^.e experimen:al cr s=acrs- s-:oked ccntro'l groups; taerefor e the sar.:e nu....,_: c: ^az::sters i1='-1 will be rec,uired. 9) Observations d_-: - a-? uccn termination of s:•_?v The fc:lo.:ing factcrs be studied dur i: g and on ter:ni:.ation o! t:-e chronic inhalation o: s-oke in inbred har::ster s. .. 'w,'ee<'ty charoes c:d wei- of the ar.i:na's from the expe--..•er.tal and fro:n the two ce-trci g: ouos. Average body K•eights for each gr oup will be plotted as a:•,:nc-icn ef ti:ae. The differences in average b=c}• weights between e:coeri.-ne-tal and control groups reflects oz the general toxic, though not n ecessarily lethal, effects of smoke inhalatior :Mdi- vidual body weights will also be recorded. These latter r..easure--er•ts are of great irr.po:tance tc•xard the end of the sr.:dy, i. e. after 60 weeks of exposure to smoke, when a sudden increase or decrease of the i=di- vidual body w•eigh: -- usually 1C;e or :r:ore within one %veei -- serves as an un.-nistakable irdicat:oL that ar. animal is moribund. - 7- I I 2 1 ~ CTR CONTRRCTS 005696 :2 11225437 C "rR HN 042~- 1116;