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I~ -'j ! HK0924001 C C~ ~Iq'I'ZAL: Ca__._nc er Cases ROLE AND RESULTS CF ANIMAL EXPERI]~ENTATION IN CANCER RES~ARCH August 7, 1959 D. J. Cohu F.P. Haas CI-R 98 COHG 0'10PP

HKOD24002 INDEX Introduction.... .... ....,...°,o...... .... • .......,.o°........ P_-~Z 1 I The Role of Animal Experiments in Cancer Research. ............ .......,............... .... .. 2 f. II AG The Results of Anlm~l Experiments ............. , .......... 7 Experiments with regard to skln and subcutaneous tissue. 1. Tobacco .......................................... 8 2. Negative results with tobacco .................... 18 a. Mouse paimtimge and ingestion ................. 18 b. Embryonic tissue tramsplants ................ ,. e. Other test animals ............................ 23 3. Tests with other substances ................ , ..... 24 B. Experiments with regard to the lung and other sites i. Tobacco .......................................... 28 ~. Inhalatio~ experiments ........................ 28 b. Direct application ...... . ............. . .....,. 32 2, Experimemts using other substamces ............... 34 C. Experiments dealing with the process of carcinogenesis ........................ ..,. I~ Conclusion ................................................... 49 ( C]"l::t 98 COI'IG 0107,9

f HK0924003 1. August 7, 1959 Center CaseB ROLE AND F~SULTS C~ ANIMAL EY/~RI~NTATIC~ IN cANCER RESEARCH The use of animal experiments in cancer research is extensive. One article attempting to collect references with regard to experiments with carcimogene lists a total of 17hO papers.1 In addltlomt the Public Health Service has published three large volumes entitled "Survey of Components ~hich Have Been Tested for Carcinogenic Activity". A selec- tive review of this literature by a layms~ leaves the impression of a confused and inconclusive state of knowledge. Not only are the results of various experiments inconsistent, but their value in relation to human beings (i.e., extropolation) is in itself a subJec~ of controversy. This memorandum will attempt an objective review of the various theories with regard to the role of experimental research in eameer and the results of such research. Accompanying this memorandum are suggested lines for interrOgating our opponents' experts, based upcm the material herein. I. Sampey, In vivo Experiments ~ith Carcinogens, X, Amer. J. Pharm.~ 305, Sept. i~56. (Our~eht Digest, IX l~'77~an. 1957) ( CTR 98 CON5 0107

HK0824004 I TEE ROLE ~ ANIMAL EXPERIMENTS IN CANCER RESEARCH ¢- The introduction to the second edition of the Public Health Service publication "Survey of Cc~rpoments which Have Been Tested for Carcinogenic Activity, 1951" discusses the use of animal experiments 2 which test substances for carcinogenic activity. "This is a convenient place to discuss o~e of the pitfalls in the use of a survey like this to affirm or deny a proposed theory of carcinogenesis or a fancied correlation between careinogenicity sad chemical struc- ture. There is a tendency on the part of some to con- sider carcinogenicity or lack of carcinogenicity as characteristic properties of chemical compounds. It is, sometimes, not realized that lack of carcinogealc potency implies such a lack only under limited experi- mental conditions. This has been well emphasized by Shear and Leiter (J. Nat. Cancer Ipst. 2_, 254 (1941)) as follows. '...it is desirable to bear im miod that the statement to the effect that given eompouods are noncarcimogenic does not necessarily mean that they are incapable of inducing maligmant tumors; it meams only that the compounds did not give rise to tumors under the conditions of certain experiments Of restricted scope. For example, the hundreds of such compounds examined for potency in Kenna~ay's laboratory in London and im this laboratory have, with few exceptions, been tested in mice only. The small number which were tested in several species of animals have given results which make clear that there are pronounced differences in the response of different species to the action of compoumde carcinogenic in the case of the mouse. Moreover, there is evidence that some ccmpounds which were negative in the mouse can induce tumors in other species... "'Furthermore, in the mouse itself~ it is now abundantly evident that different tissues respond differently to the same coa~oumd. Same eompou~d~ have ( 2. With regard to differences among species see also Simpson, Current Digest~ Attachment llI (7) July 1958; and Rigdont Hearings Subcom- mittee of the Committee om Government Operations, II.R. 1/9, 85th Cong., let Seas. 1957. L:]-R 98 CONI3 01080

J HKO924005 3. ( "given negative results vhen applied to the skin but have been quite active when in~ected subcutamec~sly. With other co~poumds both techniques have giveu positive re. sults but have brou@ht out prououmced differences in the response of the skin and of the sul~cutameoua tissue. Compounds that are l~otent for the skln end subcutaneous tissue were found to be inactive ~hen introduced directly Into the liver .... The susceptibility of the lunge... does not necessarily parallel that of the skin or the subcutaneous tissue. "'The mode of administration may also influence the results markedly. The dose may be too h18h as well as too low, and the moat effective dose for ode e~poumd is not necessarily the same as that for another closely related one. The solvent or vehicle may affect the results pro- foundly. Media of complex naturc~ such as lard or sesame oil, ~ay comtaln antlcarcinogens or co-carclnogens... and even chemically homogenous vehicles may give different results...with the same dose of the same carclnogem admin- istered to the same tissue of the mouse. "'Moreover, the sex of the animal is not without in- fluence on the results.,, . It is also conceivable that the life span of the mouse is a factor, Since come cOm- pounds ~hieh were negative during the first year gave rlae to tumors during the second year Of the experiment, it is possible that some compounds which gave negative results in the mo~se might give positive results if its life span were icu~er. Diet~ too, may he an important factor,,, • "'For these various reasons the considerations, stated in an earlier paper...lu a dlecu~siOn of the con- cept "average time" of the latent period of carcluo~ens, are also applicable here, via, the term "c~rcin~enic potency" as used in these studies Is not to be eomaldere~ ae an invariable property inherent in a cc~p~n~nd but is merely a summary of the results of particular experlmentm and is valid only for animals of the species, strai~, sexs ~e, diet~ etc., of the particular animal e~loyed, as uell as the dose, ~enstruum, tnccle ~ site of applicationt etc., of the ecx~pc~xl in questlo~ .... Comelusic~e re- 8arding the potency of amy g~Iven c~mpounde should there- fore be imterpreted i~ the lieht of the data upon ~hlch they are based.' "Amother pitfall is the attempt to carry over, ",~t,th- out reservation2 to man, cc~elusioms based on amlmal ex- periments. We do not kmow whether man is more or lees susceptible than mice to ~tlcular earelmogells. S~e animal specles~ such as the rat~ rabbit am~ dog, are ~ch more resistant to certain chemical carcino~eme than le the EIR 98 EOI, IE, 01081

HKO924006 "mouse, and vice verse, While in the monkey ncz~ of the powerful carcinogens has been shown to produce tumors. It would, therefore~ be de~geroua to comclude that man is resistant or susceptible to a given careino~en merely on the basis of experiments with e single species Of laborator~ ~ml~l since we also know of several che~Ll agents vhlch, as occupational hazards, e~e responsible for so~e human cancer and which have sO far yielded negative results in laboratory animals. Xn the light of species specificity, a given compound, whether or ~ot it is car- cinogenic to e~imale, may or may not be capable of produc- ing cancer in man. In view of the large number and variety of carcinogenic compounds, we must regard this multiplicity Of newly developed and yet to be developed drugs, dyes, food additives and other chemical compounds with which we come in personal contact as pc~sible cancer hazards, and we must reserve Judgment on specific compounds until they are adequately investigated." (pp. I-2) C See also Supplement I published in 1957, p. 2. Similar views are stated by Shublk in an excellent review of the field.3 However Shubik does feel that: "There is no way yet of decldiDg the correct species to be used for a particular carcino6enicity test, since results in the mouse~ rat, or any laboratory animal may possibly have no bearing on the human hazard. This, of course, is a co~sideratlon invoked in any animal test of biological activity, and the criticism does not apply any more to carcinogenicity than to any other form of toxicity. Experimental tests with carcinogens that do parallel ex- perience with human carcinogenesiss even to the point of yieldln8 tumors of the same morphological type at the same site, are by no means infrequent, as with coal tar, aromatic amines, xrays. It must be emphasized, however~ that these parallels are very broad in outllee and cannot give us precise imform~tlon of the quamtltatlve type often de- sired. Species variations are cc~mom in the qualitative nature of the results add even im the presence or absence of an absolute response. It must be recogalzed that moo definite ne~atlve results for the humem can.....be inferred from tests restricted to a sin~le species. Isolated posi- tive tests can be used as indicators of posslble hazards C 3. Shubik and Slce, Chemical Carcino~enesls as a Chronic Toxicity Test: A Reviewr Cancer Rem. 16, 72G-42, 195G. [ITR 88 EOHG 01082

HK0924007 5. "but are rarely adequate as the basis of hard and fast rulings. There is no reason for recommending the use of particular species except in Iostamces of compounds allied to kmowm carcinogens .... " (pP, 729-730) However~ Shubik does emphasize the v~lue of animal experiments. '~xperimental findings of carclnogenlclty do not prove that the agent In question ha~ a sLmllar role in man; such findings dot however, provide a cogent warning of a possible similar action. The number of parallels between human and animal carcinogenesis are now sufficient for appropriate use to be made of such indications. There are few who would recommend acetylaminofluorene for uncontrolled usage# and yet no hums~ data on the activity Of this substance are~ fortunatelyI available." (P. 737) Other investigators, however, are willing to ascribe greater importance to animal experiments. In a review of the smoking-lung cancer problem Cornfield et al.h admit that direct extrapolation from one species to another is unjustified. However, since the results of animal experiments are fully consistent with the human clinical, statistical and epidemiological data, they are said to attain greater status. The two investigators who seem to give meet weight to a~Imal experiments are Wymder and Kotln. Their method of interpreta- tion of the human and experimental data are similar; their only differ- ence being that Kotim feels air pollution a most important factOr, Wynder cigarette smoking. They both accept the multiple etiology Of cancer and agree that both factors play a role in carcinogenesis. Eotim'a view with regard to the role of animal experin~nts was expressed in a ( Cornfield et al., Smokln~ and Lung Cancer: Recent Evidence an~ Discussion of Sc~e Questions, J. Nat. Cancer Ins-t.~ 22 (i), 173-203, Jan. 1959. ISTR 91[ EOHG 0101:t3

b ( HK0924008 6. speech before the National Conference c~ Air Pollutio~ in Washington, November 19, 1958.5 "Certein epidemiological facets of the increasing incidence of lung camcer suggest that the atmospheric environment may be causally associated with this ob- served increase. Epidemiological data serve to indicate the areas for experix~ental biological efforts. The physical sciences provide the facilities for duplicatlng the naturally oceurrimg environment in the laboratory. With knowledge gained as a resultj laboratory biological investigations cam be properly formulated add In~le- mented. While these are necessarily primarily limited to animal species~ much slgmificant information cam be obtained. Animal studies can and do provide numerous advantages. The first and most obvious, of course, is obviating the necessity of prolonged human experi- mentation with toxic agents. Second, the use of large numbers of animals permits the study of sequential cham6es in the development of am adverse response. Careful assess- ment of host resistance factors in Overcoming these negative effects is also frequently possible. Third, protective or therapeutic measures aimed at eontrollim~ the effect of air pollutants can be quantitatively studied. Fourth, within limitst practically any state of human health from the young and vigorous to the old and partially decompensated cam he reproduced in animals.' (p. i) Wymder writes:6 'The importance of laboratory work is not to prove that smoking is a cause of cancer in -~. Such proof can only come from huz~n epidemiological imvestigation. Laboratory research can, however, contribute toj and give a logical explanation for, the human fimdlngs. Just as an animal experiment cannot disprove that a given factor causes cancer in~anbe~ause of possible species differences, ( ~e . Kotin. ExperimemtalTumor Productiom with Air Pollutants, Press Release, U.S. Dept. Health# Education and Welfa~e~ Nov. 19, 1958. In this connection Heller saw difficulties in extrapolatic~ to humams. See Air Pollution and Cancer (3)-First Discusslc~ of Dr. Mancuso'sand Dr. Kotin's Presentations p Proc. Natl. Con~. omAir Pollution Nov. 18-201 19581 pp. 232-3. Wyuder, Laborator~rComtributions to the Tobaceo-Camcer Problem, Brit. Med. J. 317-22, Feb. 71 1959. EIFI 98 ISOH6 01084.

\ C HK0924009 7. "sos by itself, an experiment cannot prove a given agent to be carcinogenic to man. It is primarily as a corollary to the human findings that the animal experiment ham its significance. The basic tasks of laboratory research# which are of a biological and chemical ~ature, are to identify the specific agents ic a given product that pro- duces cancer and to devise ways and means whereby such agemts can be reduced or removed. Im so doing, ~e cam only assume that the agents responsible for the activity in a~Imals are also responsible for the human activity. In view of the ~y similarities established for turnout 8rowth in animals and mant such an aeeumption~ thou@h it cannot be proved, stands on a firm fnundatio~." II THE RESULTS (F AN~4AL EXPERIMENTS The field of animal experiments with regard to carcinogenesis is immense. The number of substances te~ted, the species of animals used~ the methods of application, the sites of applicatlo~s the doees used~ etc. present innumerable opportunities for variation.7 Therefore this great amount of literature had to be reduced selectively to the most important articles affecting our particular problem. The initial screening has been done by Central File. Im order to present the material in more ~anagable form, a more or less arbitrary organization will be followed. Experiments with regard to direct application of the suspected carcinogen to the skin or by subcutaneous injection will be discussed first. Experiments will be divided into those using tobacco products and those using other substances. Experiments with re6ard to the ~ and other internal sirs will then be discussed, also in division c~ tobacco and mom-tobacco substances. C 7. See Shubik and Sloe, ?p. cit. supra, for the problems lnvolvc~l in setting up an experiment. CIR CONG 01085

( ( Roffo HK0924010 8. A. Experiments with regard to skln and subcutaneous tissue. I. Tobacco The first experiments with tobacco as a po~slble carelaogen date back to the early 1900's. Soweverj it was Dot until 1953 that the first significant results were obtained. Wymders Graham & Cro~iager reported io December of 19538 that CAP i mice painted with tobacco smoke condensate showed a strikingly hi~her incidence of skin cancer than un- treated mlce. The authors reviewed prior attempts to induce cancer with tobacco tar which were in the most part umanimously negative. (The article contains a table of prior experiments which is a useful refer- ence.) Roffo in 19389 reported a significan~ number of cancers om the i~uer ears of rabbits painted ~ith tobacco smoke distillate (a distillate derived from heating tobacco at 120° C for six days). However Flory in 194110 va~ unable to duplicate Roffo's experiment. He obtal~,~d some papillomas (benign epithelial tumors) and carcinom~toids (a growth resembling a cancer) in his rabbits but no true cancers. He suggested that Hoffo's chan~es were really similar to the ones he obtained and not true cancers. C 8. Wynder, Graham and Croninger, Experimental Production of Carcinomas with Cigarette Tar, Cancer Rea., 13 (12), 855-64, Dec. 195S. 9. Roffo, Unidad Caaceri~ena de 1o8 Alqultrames de Diversos Tipoe [l~-'~-Eg~-721de Tabacos, BoletinSept. de11938.Imstituto de Med. Exper. pare Caacer~ 15 FloryI The Production of Tu~ora by Tobaoco T~s~ Cancer Res.~ 1 (~), 262-76, 194i. dTR 98 COl,15 01086