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ANNUAL ItI:POItT u/ llic SCIL•'N"1'IFIC I)IIiF:C'I'OIi CLAI(ENV:F: l:OO): LITI'LF:, tic.D.

TABLE OF CONTBV'I5 ~ I ~ Page I .~ Ob.crvations . . . . . . . . . . . . . . . . . . Research Findings . . . . . . . . . . . . . . . . 5 16 IV :~ Z: Abstracts o( Reports ~ U ~'..' Grcinogenula Studies . . . . . . . . . . 29 . . . ~ Cardiovascular Studla . . . . . . . . . . . . . 32 Studics at the CcUular Levrl . . . . . . . . . . . . 43 Pulmonary Phyaiology and Anatomy Studies ...... 45 Psycho-Physiological Studies . . . . . . . . . . . 48 Tobacco Chemistry and Bjochcmiatry Studies ...... 49 Pharmacology Studies . . . . . . . . . . . . . 51 Other Studies . . . . . . . . . . . . . . . . 52 Recipients of Grants . . . . . . . . . . . . . . . . SS i`

SCIIIY'I7F1C ADVISORY BOARD to TLe Coancv iot Tobacco Raearcb--U.S.A. KFNNEIH MPRRILL LYNCH, M.D., ScD., LL.D., Chairman C/wnocllor and ProJuaor of Parholosy Medical Colkya of Sootb Carolina, Charlcstoo, South Carolina HOWARD B. ANDERVONT, Sc.D. Scienr!/ic Editor, Tbe Journal of the National Cancer Institute Bethesda, Maryland RICHARD l. BiNd, M.D. Pro/essor and Chobman, Departmatt of Medicine Wayne Sute University College of Medicine, Detroit, Michigan McKEEN CATTBLL, Px.D., M.D. ProJcsror Enttritur of Phannacolopy Cornell University MedicaJ Colkge, New York, N. Y. LEON O. JACOBSON, M.D. ProJessor and Chdrrrman, Department of Medicine University of Cbiago Dlnctor, Argonne Canocr Research Hospital, Chicago, Illinois PAUL KOTIN, M.D. .4srodate DJmtor for Field Studies National Canoa IaKitute, Bethesda, Maryland CLARENCE COOK LiTPLB, Sc.D., LL.D., Ltrr.D. Scknd(.c DJ.eetar. The Council for Tobacco Research-U.S.A. DLrcror F.mnitta, Raooa B. Jacbon Memorial Labocatory Bar Harbot, Maino STANLEY P. REIMANN, M.D., Sc.D. Director Emsr/nu, The Insihttte for Canecr Research PhBadelpaia, Pennsylvania WILLiAM F. RIENHOFF, Ja., M.D. Pro/esaor Enuritrr,r of Srorcry Johns Hopkins UnIversity School of Medicine, Baltimore, Maryland EDWIN B. WILSON. PN.D., LLD. P!'Ofp7or E/Mrft4l of Vital SlGtlSticJ Harvard University, Cambridge, Massachusetts ROBERT C. HOCKETT, Ptr.D. J. MORRISON BRADY, M.D. Axnoclote Scientific Director Associate Scientific Director I I Observations A decade has passed since the Tobacco Industry Research Committee, now called The Council for Tobacco Research-U.S.A., and the Sckntitlc Advisory Board began their work. During these ten years a major research program on the relationships of smoking to health has been initiated, amplified, and has gradually assumed clear-cut shape and direction. Research on these and related problems haa similarly expanded under many other auspices-Fedcral health agencitx, voluntary health and sci- entific organizations, universities and medical schools, and teaching and research hospitals. Significant advances have occurred in scientific knowledge and under- standing. It now seems appropriate to review the progress and the problems of this first decade, and to consider their implications for the future. Perhaps the most significant aspect of such a review is the realization that the passage of a substantial period of titne-and the aocomplishmcnt of a substantial bodyy of work-have not changed the fundamental prob- lcros. As is often the case in basic scientific exploration, intensive research has raised more new questions than it has answered; the task before us is, i( anything, larger and more complex than it appeared a decade ago, and the major research problems underlying the relationships of smoking to health, though somewhat better dcfined, remain formidabk. At the time the Board was established, for example, when cpi- demiological studies reporting an association between cigarette smoking and cancer of the lung were being advanood as a major support for the hypothesis that this relationship was pusal, the need for intensive research in many arcas-bioassay, genetip, pathoiogy, bioclxmistry and others- to explore this and alternative hypothacs, and to elucidate basic under- lying biological mechanisms, was evident. In the fntervening decade experi- mental work in the various areas has raised many new issues. The need for further knowledge (without which any hypothesis will lack confirma- tion) is still clearly evident. After ten years, the fact remains that knowledge is insufficient either to provide adequate proof of any hypothesis or to define the basic mechan- ums of health and disease with which we are concerned. It is true now as it was in 1954 that continued research in all areas where knowledge is de6cient offcn the best hope for the future. 5

Whik the substantive areas of research, and specific research results have varied widely during this decade, catain broad principlcs have emerged which serve as a useful guide to further planning. Among these may be listed the foAowing: (1) TAe Uechawring Motatre oJ Dt.ee.e C'.onceptions of disease vary enormously with changes in methods of detecting and defining them. Every new techniquc-and there have been many-for the detection, description, analysis, treatment or prevention of a disease entity tends to alter, to some extent, perception of that entity. Thus it is easy to forget (but important to remember) that the basic noture of that entity-whatever it is-remains unchanged. The nature of the intracellular biochemical process that initiates the development of a squamous cell carcinoma of the lung, for example, was the same in 1664, 1764, 1864. and 1964, despite all the changes in knowl- edge and view of it that have occurred during that time span. This under- lying consistency of the basic process must be appreciated during the search for facton which may initiate, accelerate or delay its occurrence. It is equally important to be aware that any of these modifying factors could originate, incrcase, diminish or disappear with the passage of time and with change in bio->+ocial conditioos, and that these events could in turn lead to changes in the relative frequency of different diseases. The elucidation of such factors is important in direct proportion to the extent that they throw light on the basic-and as yet imperfcctly understood-mechanisms which they atfect. (2) Tbe Distinction Between Direct Cawe, Contributing Cwrae, .lagreratlon .nd 1Von-Speclfic Aa.ocJatlon The importance of these distinctions in the search for basic knowledge has also become evidrnt during the past decade. They must influence one's thinking not only with regard to basic mcchanisms of disease but also with regard to control, prevention and treatnxnt. 'llte problems raised by conditions which have been idcntificd as "primary causes" of a disease entity (for example the tubercle bacillus in tubcrculosis) are scientifically quite different from those raised by con- ditions which have been identified as acting in certain instances, or by conditions identified as non-specifically aasociated with a disease entity. Further, the problems raised by agents which are selective in their nature (e.g. sugar for diabetka) tuo vastly different from those which are non-selective or omni-eIIective (e.g. botulinus toxin in food poisoning). 6 The latter are matters of universal eoncetn,.rhik the former focus atten- tion on the need to define the mechanisms of selectivity and the nature of the susceptible portion of the total population. Yet another type of problem is raised by factors which through association may appear to be selective but which are not eftcctive agents in the disease at all, for exampk, the cating of corn and pcllagra. Again, the distinction• between agents which act "causally" and those which aggravate or exacerbate the progress or development of a disease once it is established is of obvious Importance both to an understanding of mechanisms and to the design of effective intervention. These distinctions underlie every consideration of morbidity, mortality and prevention of disease. These distinctions have not been adequately investigated in connec- tion with lung cancer, cardiovascular or other diseases, by the types of study which can yield pertinent data, and the design and conduct of such study remains a major problem for the future. (3) The Importance oJ llost Foctor. 1n Disease Re.ponse Although the essential role of bost facton in disease response is rr:mg- nized, very little organized research to detlne and analyze these lactors has been or is being conducted in the area of smoking and health. The fact that such research is difficult, costly and time-consuming haa undoubtedly been a deterrent; one may recognize the practical inGuenoe of this situation on the individual research worker who naturally prefers to select problems towards the solution of which months rather than years, and years rather than a life-time, will contn'bute. Host factors promise particular pertinence In exploration of the problem of lung cancer, which occurs in persons who have never been exposed to the alleged causative agents, and which also occurs in only a very small pro- portion of those who have been so exposed, sq that a major scientific qucstion centers on the "why" and "how" of the detection and selection of individuals who will develop lung cancer. The available evidence demands attention to genetic, constitutional and other host factors. Such knowledge is lacking. In the development of this knowledge one must be realistic and patient. Tlte number of different genetic or constitu- tional patterns among human individuals is vast, and the distinction between most of them by any simple or convenient method is not as yet practical. They arc unlikely to be of the genetically simple type which can be detected by the methods of orthodox Mendelian analysis; they will, in all probability, contribute to the discovery of biochemical types that can be associated with relatively increased or decreased risks in a given individual. 7

a , '~'`11 '"~' ~t _ n/dle- V d0 a ttM ~ 1 ~ ^ bio~.ic~ •l+etar>rW1, a obWtt 'di+pt;` oI, dit ptexw oc Datwt.Doi.p tatlo" ir tn.uatd~ apply 9111110101011 . t>r. t...ltl.~ dtttar ,I1 rill 6 - r- rr0 the Imitations of 1~ POMWC ` ~ `by traiWOd pastonnol ob.eiratio. (..~, wbaa aoatrscy tted bat atabii.hod by the trod txiostlec peooodtt~s;~ a.atysis to tako account a ttu.y atrooottoaod .ai.ei~.. . A p.rtic.i.r ptmbit+. In tttn eacre.t apoctrntn o( research on smokiag and beakb Is tlre hck of Ittloe2 s, dkect loagtNdinal study of various types of stnaDes popdatioot tttst:mbiod and controlled, measured, tcsted and oaatvod for the origin and devoloprnent o( changes in physiobgical prooea.es oi possibio slgnibqace as precursors to, or ateps In, the origin and development o( chronic disease. Such studies require long-term eHort by highly trained peraottncl and tLay are costly; yet the order of accuracy of data from such eQotts Is likely to make them much more important than turther repuition o( less exact typa of studia. Fsstura Research 'Ibere are several ways in which the maln linu of future research may be Listed and discuwod. But no matter in what way this is done it should be rememberod that many overlapping and inta-related topics and problems will eras tho boo.darip within any attempted classification. At the pret+oot The G1QaracB fa Toa.cco Research is especially oon- oerned with the Fnhlatiat nA4 t+tsppott of rrsearch under the following broad bqdinp. A. Mot+a direct ob.arntttio~l cfldcat patboiogic studies of special controlled poptl}atlcAL . I . B. Ddlnitioa and e.abatiottt d motbods oi bioassay for whole smoke and its prodnaM, fp~ttdie~ ebtmical analysis of smoke. i f t , ea~MrdtJGClkn mikrsaakw Luft conoot• Is a ra[e f r.ctloo dcvelops loag dlseat{a Of even the tbo LssWI . only a very thoe+elaw is to disoorot wycbo-otrtotiooal) the p.j ' and aoeraook~as, sfiicoo sR dF:eCt, ob.artatloml cddolt ~ r uah ova laos poriod. aI tlaa: ; Thero ara many ways In wbklt rttw tiad suoes ptomirtn= stodbs: may be t+ct up. For exampb,)! oortaln coMdbt1 populations In htrgs medical ocntas could be nadW trequost dh.ct obaa.atloo and tests, cfocs to significant factors In the origin of eoetstitutboal or chronic dbease such as cancer, cardiovascolar disease or chronic rapitatory disease might well emerge. A pilot study along these lines Is already in progress. It may be necessary to focus the attention of any such particular expanded clinical study on one or two objectives chosen from the following: lung cancer, cardiovascular diseases, chronic respiratory diseases, psycho- emotional types. Wberever possible, honever, data on ap of the major problems should be gathered. It should also be noted that the use of twins Involving a comparison of identical and non-identkal pairs Is a valuable technique which has not been used nearly to the optimum degree. Various factors as to which data ars already available in epidemio- logical studies should also be borae In mind and should be included wherever the prospect of obtaining pertinent and ralid data seems good. Among these may be listed history of previous hrng infections, inhalation vs. non-inhalation, family history of motbidlty and roortality, occupational haurds, etc. B. D.ju.lrlow and 8odwetlow./ M.t" .j db.a.oy Some years ago the distinjuisbad biocSomhR Dr. A. J. Haagcn-Smit stated to the Scientific Advisory Board to the Tobacco Industry Research 9

i Commitme that tta derrratlva would number a aocat CbeO1lG1 fraClbGatlon rnetbod or JnOthodf of ai tbeae various xno.Io* ot h in fatx vocy avodt p40ooo saooka and - ~ . . •' a-• r,.n ~ , a...z . >r,.,A ?~r" ! w:.~. i ifJ { .• U a . -n '~ af i: afa~.tiYiiVo! aasay or for eralnatfot. As assay tochniqtta tt?l the (oa rettl jttstLlcadoO o( to o( Ws 1 OfIOCts a[~t. p[opnrrtha of smoke for bWoSical oo living anitm+la. qtt dwi.ed., Including aaays for measuring possibio "pnsFooVOkitya' bY ovett aomi-quantitativo methods, our =enoral plan haa been /bsr to measuro over-all effects of whole smoke and then, when a possibly significant biological effect has been detected, to trace it to the specific substances or properties that may be responsible for it. The spot-light of emphasis bas tberefore been consistently on biology ratber than on chemistry. In accord with this general plan, for example, The Council has con- csntrated much morc attention upon the test and trial of systems for evaluating the possible carcinosenitdty of whole smoke or of smoke con- densates peepared under very carefully defined and reproducible condi- tions, than upon the iwtation and idoatifleatioe of auch possible ingredients as polynudear hydrocarbons. 9Vblie t+everal hydrocarbons of this class have been ideatifiod in ~ttomoty amalA 3racss In cigarette smoke by in- vestiptas under otbar apqoaorahip, tlat.0 fdiottbcatioos have not answered the prime questiaa whttber sa>c1t naea' aue, aJoro the threshold of biological aj=ni6eaooa up*x t!m tscrital eopdkions o( human life. Henoe rnnoentratioa upon bio.wy tttiIl ootoorttdt ttpantion to a much greater degree thw does chemical aasbytdr. The Council for Tobacco ,R aeoaelt plans to focus major attention on critique and itank evaluation a[ ttr pteseotfy used methods of bioasuy. There would aocm b be c.etain tatiooa1 oonclusions that can be drawn in the primary so3octb.>oi 'Ioottiotft and aasay. Three of these are as fouows: '. . , :. .. h Wbar+c'ver po.sibllo, tbo oq.a or tlauo which shows pathogonic changes fn man should bo.dw tarjot orytn or tissue in the animal oaod. II. Wherever posaibie, thu srai or a=eats suspoctcd of ctioloyical patboyenic eap.city lp tiDo sito.id be appiiod to animals in the same chemical 1p[s ' as 469 in ylid" k is applkd to man, e. j., wboi. aotoka ' 10 i J t . 111. Whcrever poss(bb, too avspoctod a~eat or apents should bc appLed to ~ li is applied to tahaa. ~ : This does not ux>an ~ c~ o(,~y may not be taieltil or valuable. It emP~. > es betweea admal experiments and maa anralid apd scientific conditions which aro attaiopb1p, prl.bipt seduced to a miaimum and similarities be iacrnaod 'Op tt J§*ziMUta: 'i%p%b diQoneaoos amons animal species tested wW always praruet tofr sjtnpia a.d diroet omapolatlon to humans, data pined throuy4 tpdt 1eW pR'WY e.a aid understanding of basic biochemical processes and twLpd Cliuictal abaratlona that abouid be made directly on humans. Whenever and wherever artiAcially isolatod or prepared fractions or components of the naturally occurring form of a complex suspected agent are used to prove the physiological activity of that agent certain principles seem to apply: First; it must be shown that a similar isolation or chemical process to that which artificially produocs the derivative, takes place naturally in human smoking. Results obtainod with a norttquivalent derivative arc clearly not comparable to the actual problem In rlvo. Second; it must be aAown that any effect of a prepared derivative or component can be and is obtained by a less amount and by dosaas equIvaknt to or less thap that which actually challenges the human dssue. Higher con- centrations or larger do.cs of the challenging material create an entirely different set of circumstanees biodbatkally speaking, and therefore art not comparable with or quantitativetyttaasforttbie to the problem in humans. Third; the method of apptkat{at or contad.rlth the challenged tissue must be of the same nature as the .atural metbod of oontact 'or appl3ca- tion in humans. The elfecta of possible m.chaaical or biochemical trauma in a diffetent experimental procedure anust be toootnized as variables not naturally occurring in man. If an experimental targot organ or tissue different from that of man is selected, the reasons for the ebok.ti must be stated and explained. Such factors as greater ccxrvenLenco, economy and odrOr material advanta=es must be frankly facod, evaluated and balanced agalrut the scientific varia- bles and diBerences Involved. Factors of this sort have a strong pracNcnl appeal. Premature or erroneous conclusions drawn from unsound premises can, however, constitutc a=reat obstacle to the aeateb for the truth. No matter, therefore, bow long and arduous may be the road t6 a proper evaluation of methods of blousay and to the devdopment of a sound perspective in their Intor-telatioasltips, that rwd must be traveled. The Council intends to do its beat to aan that thia L ahe case. 11

G B'(1.arioJosy nwd Sfadatlosf .tnalyaJa It is generally oonoeded sdenti8cally that epidemiological data on mortality are not the basis for determining the origin of the diseases in which tobacco has been asserted to play a role. Such data will be subject to continu- ing study and evaluation by'Ihe Council for Tobacco Rcsearch and by other agencies active in supporting laboratory or clinical research. For example, atatiaticai findinp have suggested future research needs aiong a number of lines. I. Methods for deffaito quantitative meaaurement of the extent and degree of inhalation need to be devised in order to facilitate the detcrmination of the role, If any, which that process may play in any estimated or observed physiobgical activity of imokc. The present "quesiionnairo" or "personal opinion" method of csti- mating the amourtt of inhalation is not as accurate as would be others that aun bedriviriod. In such an Important iuue as the quaatkatioa of the d.it.od .Geets o( wpoking, the most precise aad impettonal stetbods of morawrnseat which are available are essential to sound twientibc ov.loation. ' 2 It would be impatant to determine any cumulative effects of ex- powre to amoks and whether aocamulatlon It any proceeds at a ateady aod matbomatically predictable rate. Studies are being, and should oootinoe b be, tnade to gnd wbotber any "threshold" rola- tiomhip oajsta. 3. A male excess In ioddeaae of and in mortality from most If not all respiratory diseases Is particularly marked. On what is this de- peadeat? To wbat dcgroo are endocrine factors involved in the geaeaia or dcrdopm.at d tbeae diseases? 4. 73ero is ample evidence ihat oonftujon exists on a widespread and qu+n<itstt ve2y .}goidcant scale in mortality records because of failure to distinguish primary canoor o/ the lung from secondary or mctastatic cancer bt the lung. Additional confusion exists in that lung cancer ia oot cloady distinguished from other pathological lesions which can be confused with it. These situations must be corrected before an accurate picture of causative or contributory factors in primary ltft eanoor can be obtained. S. Ahlrough the actual aurabor ut deaths from lung cancer ia itr uea.ing becutr of powhtiot; growth and because of larger numbers of petsooa if tla,.;>d#tt ago grpups, the rate of inaeaae io the ags-oorroolod 400 0 tsh bp boon slowing down since 1930. I . A This declining rate of increase, viewed in the light of actual and pa capita consumption of cigarettes duriag this period, raises a serious question concerning the validity of the simple causation theory and demonstrates the need lor continued study and observation. Undoubtedly new groupinp of data, new types or techniques of analysis and indeed new types of controlled populations will suggest and develop other problems of interest and importanee for further investigation. If the limitations of epidcmiological research as well as Its suggestive and stimulating value are kcpt In mind, itKreased activity in such studies will be an important contributor to incrase in knowledge. I). Paycho-PAyaloJosto.J Stwl{.a There have been considerabie numbers of studies in this Be3d, all of which support the belief that absotas of diffe¢tnt types and degrees may differ from each other and from tsoo+arokOCs In pryrcho-pbysiol4c.l characteristics re&ctod In habits of livins. Certain elements, inbaent in the aatme of problems of this sort, mike their solution complex arsd dif5cntt, however. This fact, while a deterrent to undertaking racarch il the area, does not oooKitute a valid rea.oa for neglecting to roooZnac the impataaoe ol tho topic or to take the sicps necessary to •ita further ao.lysis. A promising hypothesis is that types and degrees of smoking, as well as the practice of smoking versus non-.moking, art rdkctiotts of cluracterbtic reactions of the individual in his adjustrneat to his fde experience. Scientific study based on this hypothesis might And different categories of biochemical, neurobgical, psychobgical, endoctioe and emotional drives or needs which the individual experiences and which be trics to satisfy or to control by one or more uses of, or abstioeaa froati a numbes of things such as tobacco, alcohol, eoffee, tranquilizers, eacre}to, nareotics and food. Such study might also find that the heavy cigarette smok.er, the moderate smoker and the non- smoker will prove to be different In their psycho-physiobgial characteristks, as the parallel groups under the other categories of responsive behavior to life experience may also be. Personality analysis by quationnalrsa or by hy Intaviews have been made and these have provided leads which aro suggestive and which may be developed by more refirlod methods of ro.o.reb. But it is ttnllYdy that it will be su8icieat to try to eomplole tIIN analysis e( problems of psycbo-phytiul logical nature by this sort o[ opitaiotbaied evl8eoc+a, In the process of dowtfaj, reooeftsi and .oil.cdog significant dilTer_ ences in ways that can be tttadoalqod and analyaod, tbero aro r - wIn guida 13

which may Ps+obw focysod on tlto inaatan dha produood by or ~ this way one anig}tt m and iastab7ky, ` '.NI•Cift 1•. ~ ordinato ita program of retroarch t.M tboae of other inttmtod agencies. The rrsult can only be an ircrwo in taodical kaow3odge which may rY.olve questions regarding tob.ocp ta.e as well as ~aat: tpo therapeutic and pre- ventatjve nsources ol bnmW tqodio~ ~' T trd which miSM wa'darolcp iNo-• adoleaceats. AtSpstlon should be ." ..>sieh the Popstst/1 ftrom those oarfooa.ot:nt. In do"" ol stabiUty *¢pu.trol ttad hcft of oon- ci pwt Importa~oo for aduhs or It is interesting that most physical anthropological studies which attempt to correlate morphology and psycho-emotional traits have been made on adults. In the adult stage the detection and measurement of the psycho-emotional category of traits is complicated by the involved, cumula- tive ncsponse to all the complexities and changing elements of the socio- economic environment that the individual encountered over the years. Such research is useful and lntaating. It would be much more so, however, if a foundation of critical studies of children and adolescents was built under it by future research. An attempt should be made too, to find and to study characteristics which involve close correlation of mental or emotional behavior with variation in some definable and measurable biochemical or physiological pruccssas. Cotsdusion These are a few of the many scientific problems to be confronted as the research continues on tobacco use and human health. The Tobacco Industry Research Committee has been an observer and initiator of re- nearch irr this tkld uwa the period of an entire decade. The experience pined during this period should aid The Council for Tobacco Research- U.S.A. as it carries on this lmportant work. Certainly the research prob- Ians to be faced are difficuh. They arc the problems posed to medicine by the very nature of constitutional and chronic diseases. The etiology of these diseases must be aought out and identified among all the factors that affect human health from the first to the final decade of the human life span. However, the outlook for a solution to these medical problems is more encouraging today than it was when the Tobacco Industry Research Committee was founded ten years ago. New agencies have joined in the scientific eftort to understand probkms of tobacco use and human health. Recently the American Medical Association announced a multi-million dollar research effort. The Federal aovernment is expanding its own support of research in this aros. There Is every reason to believe that The Council for Tobacco Research-U.S.A. will be able to cooperate and effectively co- I 14 15

Research Findings T'bo Tobacco Indwtcy RaoarCh Committoo was formally organized 10 yeara ago to create a pr+o8raru of reiearcb ido ttmdamoatal health problems. The pwgram was a:poctod spoo6cak b deralop facts regarding the effocts ~ of tobacco ttsa A panel Of i cientiw and phyXicia" agarood to am as a Scientific ~ AdviaotY Board to t1r C,otoon" , eatabl}sblq policy, dcSnini probioaob at~ t~ tuti~. ~ ~..!~ct t~o+t F~~ ~~ -" ' award ot tacn..at TLa Coat+m atitl~,lw1~~l~i p m7 bbont«y t.cfltdoa of its own. It was daeaia+bitt at t'j omst 04 LM raearch o( the C.omml<ooe would be 8ocompWhod throogh a pt+oyam af grants-}n-aid to Independent researchers working ln their own hMitutjom. The Coatmittee'a policics and activities have eontinued under Its recent redaigtutlon as The Council for Tobacco Racarch-U.S.A. After a decade ot' support of «scarch It seems appropriate to review s+omo of the sc'tentific Sndings that have been made. Through the eod of 1963 the Scientifb Adv't.ory Board recommended almost 500 grants for research studies and The Council has in turn made awards totaling $6,078.000. The actual output sean(<iug from The Council's program has taken the form of scicntiSc papers and reports published by individual invatigators in mcdical and scientiflc journals. To date such publications have reached a total of rnora than 350 reports. It Is to these repotu "Ibe Council musi turn in attempting to evaluate the accomplishmeats of its program. Of cottrse, it ia impossible to list all of the echievctacnts of Tbe Council's raeat+ch. However, this review will attempt to dte some signiBcant illustra- tivts examples. Biowsay for Tumorigenic Action A major difficulty with attempting to deduoe a causal connection from the statistical association between cigarette smoking and the incidence of various types of cancer, especially carcinoma of the lung, is that there am a number of other noo-rausal reasons for the existence of such an auocia- tioa which can be postulated and must be excludcd before aay final judg- mtxst can be reached. Since the simp{cst explanatory hypotlxais originally advanccd was the possibility that tobacco unoke might have activity as a direct contact ur- dnoaen, a large number of studies have been sponsored in the etIort to develop reliable snd quantitative methods for asseaing atxh possible activity in comparison with that of other substaooes and mixtures that are common in the environment and whose tnmoeigeeie action Is kaown. 1. A survey of mocDankal smoktsg de.ioos was made and ooodhioos do- fiocd under which a relattvoty uniform cigarette smoke oondearsato could be made reproducibly. The ootsditioos wan so sQodtfed as to tall within the range uadot whicb wo4pal traoke Is pcoducod by hvtma clgaretm smolcess ttaofar ati pa~lbis.' Iattbntba by previous wodtert+ to the Importanoo ot oo.~Ds>~qott o1~d~ioai t+p t1t. ciotnio.l oompo.itsoa and biodogial ttdt.lty al ,pto~a ooedooNMe ,tay 1!a„e .itLttod the results of many other earbw iaw.apson. . 2. Painting of smoke condensates from eommencial dgart:tta and from cigar tobaccos and pipe tobaccos smoked in cigarette form on the skins of mice has shown that their relative activities am inconsistent with the statistical association between cancer Incidence and the smoking of cigarettes, pipes and cigars. Moreover, the high dosages of smoke con- dcnsata roquired in these experiments, and the nearly life-long treat- ment periods required for minimal tumor production, attest to the weakness of the activity of all such preparatiotts and raise doubts as to whether normal human smoking could ever produce a dosage such as to elicit this type of action. 3. The painting or fooding of potent carcinogenic substances has been shown to increase the tolerance of mice to transplanted tumon or normal skin from other mouse atraitu, which tnnsplants would be rejected in the absence of this prettc.atment. This responsc could be related quantitatively to the dosage of carcinogenic substance and thus used as a bioassay tool. Standard cigarette amokc condensates tested by this method were inactive. 4. It had previously been shown that several carcinogenic substances }n- jected intravenously into certain mice would produce htng adcnomas in numbers that were a simple function of the dose of carcinogen given. These results were coafirmcd in a study sponsored by The Council. Then standard cigarette smoke condensates, tmd from alkaloids so that they could be given intravenously in large doses, were administered in the same manner to the ume atraln of mice. No Increase In the number of lung adnamaa was observed. 5. A one-ccll animal, parmrtalwn cnudaation, has been sApwn to be rapidly killed by exposure to ultraviolet light after overnlght contact with many carcinogenic substances of tbe potynutkar hydrocarbon family in extremely small concentrations. Council projects have to- 16 17

studied this metbod extensively to refine It and develop maximum eontroi of variables. Prelimfaary measurements with tobacco smokc condensata have shown no Itxhal action for normal smoke. 6. Several studies in which mice of various strains were subjected to in- balatioo of cigarette smoko at frequent intervals during their whole life span have failed to produce aucinoma of the lung. Some inflammatory changes were fouad, rather erratically, among the treated mice, and these were accompanied by various degrees of tissue change. Such changes wets, however, reversible If exposure was discontinued at any point, ard never proceeded to invasive carcinoma even if exposure waa continued. 7. Virus infections of the lungs of such mice were found to produce much more extensive histopathologial effects than simple smoke inhalation. Virus infection followed by long-term cigarette smoke inhalation pro- duced such eflccts more frequently and at higher kvels, but without production otcancer. 8. Condensates of tobacco smoke introduced into the lungi of several animal species in the form of solutions or suspensions so that dosages were higher than by inhalation of normal smoke have also failed to produce carcinoma of the lung. 9. It has long been known that feeding urethane to strain A mice by mouth will produce pulmonary tumors. Addition of nicotine, smoke coodensatea or tobacco smoke inhalation was found in a Council study not to enhance this effect. A further study discovcred that the vitamin niacin has a defeasive action against the urethane tumor production eHecx and that more tumors arc produced in niacin-defieicnt animals. However, even in niacin-0eficicnt animals the inhalation of tobacco smoke did not increase the production of pulmonary tumors following urethane foediag. 10. Sprajue-Dawky male rats, fed dicta containing 2-acctylaminoAuorcne and also exposed to cigarette smoke inhalation or injected with a to- baooo smoke ooodensate In peanut oil, experienced a delay in the devel- opmeat of liver changea that are considered to be indicative of a pre- canoer%xn state (gross liver damage and lowering of riboflavin content). Another condeasate, prepared by the standardized method referred to elsewhere, did not affect either the rate of cancer induction or the final tumor incidence in rats fed either 2-acetylaminoAuorepc pr 3'-methyl- 4-dimethyl-amlao. 11. It has been shown that small doses of chemical careinogens, which alooe would not produoo cancers, will do so when injected into male Swiss mice along with small do.es of certain common viruses, which i i also do not produce cancer alone. TTie investigator showed that a com- M bination occurs between the virus and the chemical substance ind poa- tulated that the virus was able to carry the chemical into the interior of the cell. Immunization of the animals against the viruses prevented the cancer production. Etiorts to show a similar binding between these viruscs and cigarette smoke condensate were unsuccessful. 12. Study of the sebaccous gland suppression in mouse skin by potynodcar hydrocarbons has sholvn that the usefulness of this phenomenon for= bioassay purposes is limited by the occurrence of false positive and :' false negative reactions and by its relative Iaseasitivity to small changes ~' in concentration or activity of the substances tested. V 13. Cigarette smoke condensatea in solution were appiied to the cervix and upper vaginal area of more than 200 DBA-1 mice. 'Ibirty one percent of mice painted twice weekly developed tumors.'Itte solvent applied alone at a similar dosagc was followed by tumors In 60% of the mice and treatment with a dry cotton applicator by tumors in 42.8%. 14. Animal and human lung cells have been successfully maintained in tiuue cultures by several Council gnntees as a basis for development of other possible methods of bioassay. In tissue culture form,ihuman cellular material can be used for testing. Satisfactory and interptntable methods for using such human cell material as a bioassay system for carcinogenicity have still to be developed. 15. A number of other possible bioasaay systems have been tried or an; under study with various degrees of success or promise. The overall impression gained from these many b'toassay studies is that tobacco smoke is inactive or of extremely minute activity In these several artificial systems, none of which of course actually measures human rtsponae under realistic conditions. The results lend little to support the hypothesis that cigarette smoke can act as a direct contact carcinogen in the human lung. Study is continuing with respect to other hypothetically possible modes of action. Ciliary.lctivity and Mucus Flow A Council study undertook to evaluate the relative importance of the activity of the cilia in the total machinery of lung clearance In intact animals since it had been reported that cigarette smoke, like many other gases and acrosols, diminished the activity of these hair-Uke appendages which coat the upper respiratory tract and which normally propel upward the blanket of mucus which carries inhaled dust and debris out of the lung and it had been suggested that delayed elimination of inhaled dust and debris, contain- 18 19