Tobacco Documents Online

PROJECT 6900 PHYS[10LOGICAL STUD~TES SE~$1 A~NUAL REPOF~T PERIOD COVEREDBY REPORT~ May, 1!966 through October, 1966 DATE OF REPORT': OCtober 25, 1966 RESF0~$SIBZLITY FORITHE PROJECT: P. C. Luchsinger OBJ:ECT~E The objective of Pro:ject 6900 is to ad~ilse Philio. Morris in biio!ogz as relased ~o smoking a~d smok:ing p~cducts. Th~s advisory ca~aclt7 3nc!udes :'in house" eonsuii%a:~ion in the design and performance of biological projects, as well' as the smpervis~on of b~ologlcal researcc related: to smokin~ in outside l!abora~ories.. More imporZ~n~ than, elther of these two actSvi~les Ss the a,c:quisltlon of information which may be used to, formulate future pro:~ec~s relazin~ to smoking and heal, th. Because of this. la~st acti!v:ity, it seems appropriate to review, briefly, She philosophz underlyPng the biological s~udies. eilther carrie~ out under or endors~ed ty P;oje~t 6900. These s~u£ies may be divided into. 3 categorles:~ (I) StudSes relapsed to smoking and disease en~ties, (2)i Developmen~ of tests ~o be used in product eval!ua~t~on and production (3)' Basic: research rela~ed to, ~moke deposition and absorption. I. Studlies Re~a:~ed ~o Smokln~ and Di:zea~e En~t~tie~ Tradi.tionally, the p~ob!ems of smoking and health have been rel, ated to carcinogenesis, emphysema, bronchitis and~ cardlovascular d~isea~se. Most pub~ic attention, ha;s. been~ directed~ to carclnogenesii~, pa;rtlcu&arly lung cance~. However, in terms~ of epidemLology, it Ss only of !im~ted sigmlficance. The research going on, in carcinogenesis of tobacco products ~s primarily directed toward;~skim painting and inhala~i~n studie~. In bo~h type~ of st~dle~, the German and British effort~ are large and well orga~Szedl. It wou~Id be inap~roprilate to attemp~ to duplicate ~hese extremely costly ef~ort~ In re~earnh. Not; t,obe taken fr,om this room

PROJECT 6900 P~ZSIOLOGIChL STUDIES SEMI ANNUAL REPORT PZRIOD~COVERED BY REPORT': May, 1966 threu,gh October', 196o DkTE OF REPORT: October 25, 19'66 RESFONS!BiLITY FOE THE PROJ~ECT:: P. C. Euch,singer OBJECT~;E The ob:ject.ive of Project 6900: is to advise Ph,iiip Morris in, bio:~ogy, as rela%.ed~ to smoking and smoking p:rcd~.cZs. Th~s advisory capa:c.ity imc!udes "in h,ouse" consultation in, %he design, and~ performance of biological p,roJects, as wel~ as the sug,ervision of bio,logica.l research related to smoking in outside laboratories. Move ~mporta:n5 than either of these two activities, is the acquis.~t.ion of information whic'h, may be used to fo.rmu,]a~e future projects, reTa%Ing, to smoking and~ health. Because of this. last a:c.tivlty, ~t seems appropriate to. review., brlef~y, the philosophy underlying the biological studies either ca.rried~ out umder or. endors.ed ty ~roject 690:,0o These studies may be divided into 3 categories: (i) Studies related to smoking and disease entities, (2) Development of tests to be nsed', in product evaduat~on and produetlon (3) B~s_c re:search related to smoke deposition and absorption° I Stu~dies. Re~ated to: Smokin@ and Dise.ase Enti~°= Tradit~onaily~ the problems of smoking and health have been related to carc±nogenesls., emphy.sema., bronchitis a~n,d cardiovascular dls.ease. Most publ~c atten:tiom has b,een, directed to carcinogenesis, particularly lung camcer~ However, in, terms of epi~dem~oIogy, it is only of limited significance° The research going on in carcimo,g.enesis of tobacco p:rod~u,c%s is primarily directed towards skin paln,ting and~ inhalation studies. In both types of studies, the German a:n,d Brlti~h, effo~t~ are large a~nd _ well organized. It would be inappropriate to attempt to duplicate ~hese ex%rem,ely costly, efforts ±n r~ear~h_ 000341401. For Re erence Not to: be taken from this room

~owever, it is felt desirable to: complement these vast industry wide efforts with small experiments, designed to characterize the smoking product.s c.f' Ph$1~!p Morris in terms of skin o:r lung tumor activity° At the same time it has been decided that Project 6900 will not underwrite extensive developmental, studies for new carcinogenic test~ but rather' fol~ow closely the ~ev.e~opment of' ~h.e:se teats by repeated conta.cts with lea.dzn~ investigators, in the fSeldo Based~ on this con~ideration~ ProJiect 6900 has not used any pro¢o.zoan or tissue culture te~ts~ nor ha~ it engaged in t~e study o£ the mutagenic activity of smoking products° However~ It is. fe~t that a !imlted~ exploratory effort may be ~us.tlfSed in skin hFperp~asSa as we~l as the neonata~l test, because both of these are well under way to b:ei~g recognized, as useful tests in the ev~luatlon, of ca,rcinogenlc a~tivity. Emphysema and bronchitis are the leadin~ causes respon,sible for In,capacity for work and ga~$nful empl!oyment nex% to. cardiovascular d~zeases. The ~ocia! ~ecurlt~ agency has. in.dlcate~ that ~hese two dlsease~ are mo.~e co.stly t~an any other diseases. BOt~ disease~ are believed to be closely rela~ed to air pollution. ~na.smuch as the p~obabi!Ity ex~stz tha~ these di~easez w~ll gain increasing public recognition and since cigarettes will most likely be implicated as one of the cau,sa~Ive agents in ~~he~e diseases, ~t is. felt that emphasis shou.~d be put on research in, this area~. Presently no. deflnltlve experimental animal models for studying these diseases have been developed. N!either Auerback'~s nor Anderson's studies in ~o~s have demonstrated a qu.ant~tat~ve relation$h~ip of smoking to these diseaseS:o It is therefore planned flr~t to establish a ~uan,titative hlstopa~hologlcal technique to study the diseases, and secondily to: documen,t on: a quantitative ba~i~ the relationship: between smoking and the lun.g pa.thology of these diseases°. ~ hlstochemical or bfoch.em±cal approach. might be considered only after a quantitative re~ationshlp between smokingl and bronch~Itis and/o~ emphysema has been ¢~ear~y d~emon,strated. Although i~ ~s recognized as a prominent problem by med~!cal authorities, the public ~s not fully aware of the relationship between ca.rdlova~cu.lar df~eaze and cigarette smoking. Chronic cardiovascular disease is unique to humans, certain prSmates, pigs and birds. Its relationship to: smoking may~ become more importan.t in the future° Currently, very little basic investigation of the~e diseases, as related to smoking is being carried ou,to The problem: however is of suff$cient importance that it shou,ld remain under consideration. 100034140Z

For the presen~t, ProJiect 6900 will keep abreast of the developmen~ts in thi~ area~ rather than pursue active research. Deve!opmen~t. of Tests to be Used in, Product EVaiua- tion and Production A: biological program, re!a~ted to certa~In disea~ses and cigarette smoke should be complemented by. a number of tests which may be helpful ~n the d:evelcpmen,t of smoking products. Therefore, Project 6900 considers, as one of its objectives th,e d~eve!opment and assis.tamce in, the development of such tests° The number of such tests which, can, be devised, d~esigned and exploFed: is almo.st limit1ess° The follow.ing guidelines have therefore been es.tab!ished:~ I)i The tests, should be fntu$tlvely related to. diseases allegedly related to, smoking. The tests should be rapid. The ~ ~- ~e~,~ s.houZd be highlz discr'imlna~ing between, va.rlous, smoking products.. 4)~ Te~ts shou,~d be r~l~iv~ly, inexp~emsive. A number of tests have been developed and are ~n use whd!e others are ~n the d.evelopmen~t stages° Basic Researea, Re~a~ed to Smoke Deposition, and~ Absorotlon The biological program of a c±garet~e manufacturing company should inc!ude those basic scientific studies wh~ich are a natura~ extension of its other researc~ effor~So Such research ma~y encompass studies of smoke depcsltlon, nicotine andl carbou monoxide absorptions, an~ nicotine excretion~ e~c. Such studies, can be accompi~shed using the resources of Philip Morris Domes,tlc and ~nternationa~lo S~.mmar!zing ~he foregoing, Phil~p. Morris' has taken~ a th,ree-p,ronged approach in the area og smo~in,g and hea~th. Th,e first is re!a~ted to. long-term biologica~ effect of smoking, products and encompasses the fo~lo~ng experiments: Long-term mouse skin paint~n~ Long-term primate inhalation Long-term in,hala~tion s.tudies in. rod',ents -3-

t The second concerns the development of tests for prod~c~ evaluation and production and~ encompasses the followin,g:: Mouse irritant test Short-term mouse skin pa~n~ing test Mucociliary activity in the cat klveolar macrophage survival stu,dies Clam, gill cilia inhibition stud±es The third concerns studies on the d:eposltion, retention a~nd e!im~nation of cigarette smoke and its constituents from, the orgam±sm,. They ate implemen~ ~ ~e_ in the following studies: Carboxyhemogicbin: accumulation in blood DeZermimaticn, of nicotine and. nicotine me~abolites ~n blood and urine. RESULTS Lon,$-Term Mo~Jse S~£n Paint~n~ The pa, inting of the va.rious ma~terials on, the skin has been discontinued: after 18 months.. The mice that remain will be observed through 24 mon,ths (Apr~l, 196T) for tumor formation a:nd mortality. In Table I is presented~ the data accumulated up to October, 19.66o With th,e exception o~ group ~0 in which th,e material painted had B(a)P added, It can be seen~ that the various materials from the cigarette smoke pred~ced~ very few carcinomas. A~though it will be: six months hero.re a final assessment can be made, to date the number of tumors observed is comparable to the study conducted by the British, Research, Council (Harrls.~ personal communication) but ~pp:emrs lowe~ than Wynder's re~u,~ts. Eon~,Term Prima, te inhalation Smoke ~nha~lation has been completed and all animals hmve been sacrificed:. Th,e gross exam~na:t~on of the lungs indlcmtes some: emphysema in the smoking group, even: though the physiological measurements do not reveal any differenc.es between the smokers a:n,d the monsmokerSo For the: quantitative histopathologic assesm- merit of bronchitis, the Re~d Index is being used° ~or the assessmen:t of emphysema, it has become necessary to develop a method. 1000341.404

This is cu,rren,tly, in progress and a photographic technique using whole lung lobe sections seems mo~t promising. From a qualitative standpoint, ou.r experiments indica:te that the changes in, the lung a.fter smoking are less severe than thos.e observe~ by Au,erbach. Th~s is most l~kely due to the physiologic method of smoke a~dm~n,~stration, used by Auerbach. Inhalation Studies In Rodents This sturdy has been originated in Eu:rope to complement ou.r own primate s.tud~y. It permits thee exposure of greater numbers of an,ima~is over a greater percentage of their life s.pan~ All th,e inhalation, studies have been amplifled~ by carboxyhemog~obin determinations and in the future there will also be nicotine determinations in the blood and ~ung tissue. To date no results are avai~o!e. Mucociliarv A'ctivit7 in the Cat Because of the la:ck of orecis±on in the acute studies at AME La:bo~a~to.ries, it ~as d.ecld'ed to reestablish this techniqu,e at FDRL. In Tab:le I! are the resu.lts of a~ study designed to evaluate the effects of variou,s c~garettes on mu.cociliary activlty. Using. thrifty-six cats (necessary to meet experimen,tal design, requirements for four' cigarettes), we have demonstrated th,at (in the order of decreas±ng effect) Co~mander,~ Marlboro 8!5, Ph~ilip Morris Filter and Triple Fi~%er have different effects. on mucociliary activity° The differences have b.een, establish,ed with a very h~gh degree of significance° Due to d~ifficulties in the maintenance of window cats,~ the studies could not be continued. Before proceeding furth.er, assurances a~re bein~g sought Srcm our contracto~ o~ the ma~InZenance of the test animals° Another means of accom~iishlng this obJiectlve would be to carry oust thee work "in house.~' Mouse Irritant T'es.t Th~is test measures the acu:te d:ecrease in the resplra~tory rate of mice wh,en they are exposed to c~garette smoke. In, prior work with, th~s technlque it has been shown theft differences between cigarettes can, be demonstrated:o SuDsequently we have tried to demon,strate the sensitivity of this. test. on a statistical basis. Th,e results In~ Table lil indicate that the following d:Istinctlons (at the 5% level of slgnifican,ce): can be made between the whole smoke and the gas phase o.~ four ciga~rette bra.n.ds. Th% decrease in the respiratory rate produ.ced by Marlboro 85, Commander an:d Ph±]iD Morris Filter are not differen,t from each: other. ~000~4~40S

The same is true for Triple Filter a,n,d Ph~!ip MOrris Filter. Triple F~Iter does, however, produce a slgnif~cantly different effect than Marlboro 85 and~ Co~mandero Short-Term Skin Paintin~ This is a ra~pid test (7-10 d~aFs) in which it is possible to differentiate between carcinogenic, and~ noncarclnogenic chemicais~ uslng the skin and the sebaceous glanCs Gf the mouse. The parameters measured are the ~ncrease ~n thickn,ess of the skin and the decrease in the number of sebaceous gland's. Prelimlnary t~ials with three carcin,ogenlc and th,ree noncarcinogen,ic substances indicated' that this test w~ould not be useful. This dec~sion~ was. based on th,e occurrence of two false negatives and wide varlance im~ the resu~its (Table i-V). A subsecuent trial using BIa)P and a modification, of the techn~ique indicates that this. ~echnique deserves further investigation ~Table ~)~ Clam Gill Cilia In~ib!tlon Studies and Alveolar Macron~aee Surviva~ Studies Both th,ese tests have b,een~.developed an~ t~eir objectives are nursued in collaboration w~th Project 6900o T~ey are~ however, carried out by Project 0108: a~n,d their status has been reported by Project Basic Research. Related to Smo~e Deposition: and' Absorption, At present the necessary techniques for n~cotlne determina- tlon in blood and urin,e are being developed in: collaboration with the Analytical Services Division. CONCLUSIONS AND PLA:NS Lomb-Term Skin Paimtlng The studies are progressing sati~factorilyo Fina~l resu~its are not expected', before April, 1967. I~ is recommended tha~t Philip Momr±s perform long-term skin painting studies on all new marketable ciga~rette products. ~00~~O~ Lon$-Term Inhalation Studies Available ±nformatlon from these studies indlicates that gross lung path~ology can be induced by smoking cigarettes. However, th,e degree of these changes should b~e in,terpreted in terms o@ the total amount of smoke administered, it is felt that such studies shou.ld be intenslfied~ and that the quantitative relationsh.ip between the number of cigarettes smoked and the amount of lung pathology induced be emphasized. -6-

Development of Tests to be Used~ in Prod~,ct Develop- merit and Production These tests should prov.~de a sa~tisfactory basis for the production a,nd d~evelopment of a~ new ciga.rette product. At the present time it ~s not plann~ed to develop any add~itiQna! tests, however., test refinement will be performed w.here necessary. Basic Resea.rch Related to Smoke Deposition and Absorption Development of methodology is ~n progress. The formula~- tion of the research protocol w:~l! depen,d on the complexity of the a,nalFtical pro~cedure. -7-

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TABLE II Bra~n,d Commander Marlboro 85 Philip Morr±s Filter Triple Filter Mean Decrease in Mucus F~ow,cm/sec. 2.42 2 .O9 1.9o O. 52 - 91 -