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1 I I BIOASSAY, CARCINOGENESIS and TISSUE CULTURE Dr. Reimann Date: August 18th, 1967 DDr. Loosli Application For Researc6 Grant five year plan. Dr. Jacobson #456 is concurrent on a OP 1 1 N Dr. Sommers, Chm. Ig'M' ; 0 7 #224 - 1955 - 1902 mc1. SucCFSSOR TO THE and.•enewed annually thryu _ TOBACCO IIVDUSTRY RESEARCH CO~VthfIYTEE October 1; 1966. '` yv'- . ~~qh,• If! ~ COMt+II]TTEE: 03s irulzv avr:~~u~: Preceded by #29B, #2].2 & E V N Y 0 / 1. Name of Investigator: Freddy Homburger, M. D. President and Director Bio-Research Institute, Inc. 9 Commercial Avenue Cambridge, Massachusetts 02141 4. Project or Subject: BIOASSAY OF TOBACCO SMOKE CONDENSATES AND RELATED PROBLEMS 5. Detailed Plan of Procedure (Use additional pages if more space is required.) INTR ODUCTION It is realized that our current grant from CTR has been made for one year without further commitment in order to enable us to conclude a five-year program initiated late in 1962. The present application is to request the Council to consider support for another five-year program, based upon our past performance sum- marized in the progress report. The three major projects for which we seek long-term support are the following: 1. Acceleration of growth of chemically induced tumors for the purpose of developing rapid carcinogen testing methods. 2. Systematic study of inhibitors of chemical carcinogenesis with the aim to neutralize alleged carcinogens contained in cigarette smoke condensates. '~ ' # . . , Cf 33 THB COUNCIL FOR TOBACCO RESEARCH - U.S. . Activated on 1011/62 .:..i.::: ~~.. ..: .

Bioassay of Tobacco Smoke Condensates and Related Problems conditions the decline of carcinogenicity and of co-carcinogenicity for mouse: • "skin that appears to have occurred since 19'60 in cigarette smoke condensate. entific knowledge and will contribute to the technology necessary to formulate ``;~ . ,•,~ 3. Skin-painting studies in mice to measure under cornparable '' Each of these projects is based on many years of experience in the particular field and clearly promises to yield valuable fundamental sci- cigarettes that will produce smoke condensates incapable of producing cancers when paintea on the slein oi mice. 1) Acceleration of Carcinogen Testing Th ff d i l ere are e orts un erway n many aboratories to develop rapid screening procedures for the detection of carcinogenic substances. These range from in vitro tissue culture work to the use_ of neonatal mice, newts and~ other `~`~ff'Kr tion sites represent the first significant step in this direction. We believe that it will be possible to obtain even. shorter times of' latency than so far possible by extracting from the initial carcinogen-injection or -application sites large numbers of transformed cells for transfer into fresh hosts, time to make it practical to detect even weak cancer-causing chernicals. Our studies on the transfer of multiple pooled carcinogen injec- light for paramecia and other biological phenomena have been, correlated with carcinogenic potency. However, the most reliable carcinogenesis test would still be the production of tumors in, a mammalian species in a sufficiently short species. The destruction of sebaceous glands and the increased lethality of U. V. Initial experiments on this subject are. already und'erway. The subcutaneous injection sites of C57BL/6 mice are excised after 3 to 5 weeks of contact with carcinogen (benzo[rst]pentaphene is being used as standard car- cinogen) and after mechanical dispersion by means of Snell's cytosieve, the cell suspensions are centrifugated in Ficoll®, a neutral high molecular dextran-like polysaccharide of low osmotic pressure. The cells are thereby separated ac- cording to their specific gravity and the various cell layers, some of which will contain concentrated amounts of malignant cells, are injected' into fresh hosts. By this method, it is possible to inject into a single mouse many times the number of transformed cells coming from numerous induction sites. -Based on the studies of several authors using transplanted tumors and confirmed by our own work, the larger the number of transferred' malignant cells, the more rapid the growth of tumors. It is believed that it may be possible to get a 100% tumor yield 2 weeks after transfer into new hosts or 5 to 7 weeks after the car- cinogen was first injected, 1003546825

.Bio-Research Institute, Inc. Bioassay.of Tobacco Smoke Condensates and Related Problems in carcinogen injection sites in the subcutaneous tissue five weeks after in ~' .4. jection of carcinogen. By transfer of pooled multiple injection sites, we were ~observing changes in from 2 to 3 weeks after carcinogen injection but not earlier. Thus, the shortest theoretically possible test period would be 4 weeks In the mouse, the first cytologically malignant cells are found In hamsters, on the other hand, Nettleship and Smith (Proc. Soc. Exp. Biol. Med. 74:800-802, 1950) showed morphologically transformed' fibro- possible test period wouldi be 2 to 3 weeks. I transfers could be made 1 or 2 days after injection of carcinogen and the shortest trates, will produce palpable tumors in a shorter period than in mice. Such that Nettleship's observations can be confirmed) that transfer of multiple injec- tion sites in hamsters and even more so, transfer of transformed cell concenl-. cutaneously induced tumors -is the same as in mice. It is likely (assuming blasts 24 hours after injection of methylcholanthrene. Hence, it would appear that the subcutaneous tissue of hamsters would lend itself even better than that of the mouse to carcinogen testing. The times of latency in hamsters for sub- and tumor cells transferred fronn, females into males could be identified as be- longing to the original female host and hence as induced by the carcinogen in- jected into the first (female) host. In addition, chromosome studies are readily feasible in hamsters Institute of Technology, and to carry out the above described studies. Dr. Janis Gabliks, currently Associate Professor of Cell Biology at Massachusetts culture laboratory under the direction (either part time or later on full time) of . We propose to establish during the next 2 to 3 years a tissue .transfer large numbers of cells exposed to carcinogens in vitro back into ham- ster cheek pouches. In this way, it may be possible to obtain tumor growth even more rapidly than is possible with in vivo systems alone. ' In addition, it will then become possible to extend the work of Berwald and Sachs on transformation of hamster fibroblasts in vitro and to serial transplantation. We are quite confident that these techniques applied to subcutaneous tissue, epidermis and lung tissue of mice and hamsters will make such pro- cedures as mouse-skin painting obsolete and replace them by carcinogen tests lasting less than two months and having as endpoints histologically demonstrable malignant tumors, the truly neoplastic nature of which can be ascertained by 1003546826 • While these studies will initially be done wit'h, chemical carcino- gens (strong, weak and intermediate), we shall soon be able to apply them as

Bio-Research Institute, Inc. C Bioassay of Tobacco Smoke Condensates, and Related Problems tion later this summer and condensate will be available in adequate amounts. ~ Bio-Research Consultants, originally scheduled for this spring, will be in opera- well to tobacco srnoke condensates, since the condensate producing machine of found that oxidative derivatives also have this effect. 2) Systematic Study of Inhibitors of Chemical Carcinogenesis . While Falk and Kotin showed that reduction derivatives of poly- cyclic hydrocarbons inhibit the carcinogenic effect of these hydrocarbons, we the di-quinone should be more active. corresponding to the quinones which, if the peroxide hypothesis were correct, would not possess the chemotherapeutic activity of the quinones. Conversely, they possess chemotherapeutic (in contrast to merely carcinogenesis inhibiting) effects. The hypothesis that hydrogen peroxide, which may form from quinones, is the cytotoxic agent is susceptible to test by synthetizing the aza derivatives The quinones appear to be a group of special interest because neutralize the carcinogenic effects (as tested by our own new rapid methods and by skin painting) of cigarette smoke condensates. It is the purpose of a systematic study of derivatives of benzo [rst]pentaphene, of other polycyclic hydrocarbons and of terpenes (such as li- monene) and their derivatives to find those compounds that are most active in counteracting the carcinogenic effects of polycyclic hydrocarbons and that are themselves least carcinogenic. Such compounds could be used eventually to which are known to be precursors of the cytotoxic quinones in vivo. test the trifluoroacetyl derivatives of some hydroquinones and their glycyl esters, In order to render these compounds more volatile and better suited for use as adjuvants in the tobacco blends, it is suggested to prepare and IV -_... I ...._- -1- ,a :v~ . These compounds are represented by the following examples: ~A . 0 1. 9, 10-Phenanthrene hydroquinone di~(trifluoroacetate), IV © 2. 9, 10-Phenanthrene hydroquinone-bis-trifluoroacetylglycyl `~ 3. ester, V 3, 4, 9, 10-dibenzpyrene-5, 8-di(trifluoroacetoxy), VI y~ V VI

Bio-Research Institute, Inc. Bioassay of Tobacco Smoke Condensates arnd Related Problems for the volatilization of amino acids (see, for example, Cruickshank and Sheehan easily, and the Trifluoroaceti~c acid~ and its derivatives are completely non-Jtoxic~ Anal. Chem. 36:1191, 1964). The trifluoroacetate group is known to split ver- and physiologically inert (see "Toxic Aliphatic Fluorine Compounds" by F. L; Pattison, Elsevier Pub. Co. , 1959', pp. 20;27;62). Skin-Painting Studies in Mice genicity of smoke condensates, and Wynderts early work, for lack of a better method, as a measure of carcino-' Skin painting in mice has been used since Croninger, Graham control for a comparison between mouse carcinogenicity of condensates of 1960 in 1960 of cigar tobacco which have been preserved and which could serve as a", earlier (more carcinogenic) experiments. We have some 20, 000 cigarettes made prepare at the time of his latest study condensates from cigarettes used in his W'ynder and by Bock. However, neither of these authors was in a position to duced carcinogenicity of cigarette smoke condensates have been published by originally studied by Wynder, Kensler and ourselves. Such observations on re= . ;.,,~...;,..~ This approach is based on methods used in gas •chrornatography There are indications that present day cigarettes may be less car-~. cmogensc an ess co-carcinogenic in terms of mouse skin response than those ,, zf{ and' of 1967. The evidence which suggests such an experiment is shown in Table I, summarizing our own mouse-skin painting experiments using conden- sates from various unfiltered cigarettes done in 1960, 1963, 64, 65 and 66. •..yR There is a strong suggestion here that the mouse-skin carcinoizenicity " has declined and, even more striking, that co-carcinogenicity has practically disappeared6 While the cigarettes used were the same brand in most of these studies, the source of the condensates, the machines used for smoking and the handling of the condensates were different. For these reasons, the results shown here are only suggestive and not conclusive. We are convinced, however, that a repetition of our earlier study sponsored by CTR in 1960 to 61 would yield similar and conclusive evi- dence if indeed the composition of present day cigarettes has changed. We are 1003546828 Condensates could be prepared by Bio-Research Consultants in an identical mariner for all cigarettes smoked. Condensates would be diluted with equal parts of acetone as in our previous studies, planning the experiment summarized in Table II.

Bioassay of Tobacco Smoke Condensates and R elated~ Problems By use of the ATC cigarettes (which we obtained from Dr. • 40 experiment), we could obtain a check on the reproducibility of' the first (19'60) ;. ~. Hockett and understand to be made of the cigar tobacco used in our original 1960 experiment and thereby determine that CAF1 mice respond today in a manner. , similar to 1960. We could by this device obtain a comparison with the Millerton 7 mice used by Wynder in most of his published studYes. Inclusion of the two most " ..:.:~ widely smoked non-filter cigarettes of today is logical, condensates. croton oil as a promotor yields early information on the carcinogenicity of the . The use of primed animals having received 400y of benzpyrene as initiator measures the co-carcinogenicity of the condensates, and the use of ment. It will provide conclusive evidence not obtainable by any other means and This is an elaborate, complex and protracted (two years) experi- carcinogenicity of present day cigarettes, might well demonstrate the absence of co-carcinogenicity and greatly attenuated come may be predicted! as likely. In view of our latest skin-painting studies (Table I) such an out-

. _ able I SUMMARY OF SOME RECENT ' MOUSE SKIN PAINTING EXPERIMENTS 1 ~ Date of beginning of project 1961 -62 . Jan. 1963 May 1964 Mice 0 CAF-1/Jax Millerton Charles River CAF-1/Jax Millerton , 92 Swiss yg Swiss 29 ~~ Swiss Q$ Millerton~) Swiss eg ~ N Primed with benzo[a]pyrenet - - + + A 9 "5 ~rll Cigarette condensate applied• 0 ' j Per cent tar 50% 50% 50% 18. 6% ' 50% ' 50% ` 18, f9Jo 50oJo 18.01 % 5 ^ t ~ 1 Per cent water -, - 15.4% - 15•4oJo - . ~ ~ No, of mice per group at start 100 200 45 200 150 100 50 50 50 50 00 y c;60 Papillomas after: . _ . . i . . Y .~~ . 30 weeks " 56%' 0. 5% 14% ~ 40 42 " 1% 10% ~' 58% ~ 8% 0% 0. 2% 0: ~s '~v 57 " 9% 1% a 66 " 20% 39% 6% 0. 7% 76 " 32% r 60% 12% 21% 7% ~sc f Published J. Nat. Cancer Inst. 31:1445, 1963. 4001Ag per mouse. .~ ocsftscoo-r , :;. . July 1964 ,. April 1965 Dec. ` 1965 Feb.' 1966' . P.uguet;-196E ~ . ta+:".'t~~ai; ~rr . Q

ME ® Group No. 10 11 12 Total 950 :rx+-V" ~ Application Th Council for Tobacco esearch-U.S.A, s. Bio-Research Institute, Inc. Table II ~.. • s/ . ! . . . . . r,J ~ +~ . t PROPOSED EXPERIMENT FOR CTR MOUSE SKIN PAINTING Strain of Mice CAF/1 0 No. Mice per Group 100 , " 100 Millerton Sw~ss 100 50 50 50 50 Primer (mg per mouse) 0.4 0.4' 0.4 0.4 Treatment of Mice Cigarette Smoke Condensate Derived from ATCt Brand A Br:~ne g none~ ATCt ATCt Brana 13 none # Croton Oil 0.75 d 0. 75 ~ 4 * Benzo[a]pyrene #* All cigarettes of regular length (70 mm) t All-tobacco cigarettes # Acetone control

rt Y5 ~ "6. Budget Plan: Salaries Expendable Supplies Other Expenses Permanent Equipment "" " Overhead (15% of a, b, c) 4 2/1/68-1/31/69 :~ Anticipated Duration of Work: Five years f Based on current level of endeavor._: ~ , t,... £;~8 Facilities•and Staff' Available: The same personnel as in the past will remain active in '~ "this work," In addition, a new group is being developed for studies on tissue , ~ 3 ~E ~~~`cultures" and hamster oncology in our new Cummington Street annex, It is ~ anticipated that with~ addition of increased staff in the new division for work with hamsters, some of the mouse work will be reduced so that during each of _~'X the next five years, the budget for this project will remain approximately the . ~ . _ . . . ~ .. _•;. . ~ : ~:-: : •. ~.., : ..~.; ' . . . .. ...... .. . ..'.:~ .. ..• -,:r- ~'."~F;i! .~ -. . . , _ . . , , . . .v. ..-. .. . . _ ~ . .. ~: - . .~- : ... + . . . . 2. .. . .. ~ . . n Additional Requirements: Condensates for the carcinogen inhibition and carcinogen ~. acceleration studies will be provided by Bio-Research Consultants free of charge for labor costs only because their smoking machine was developed under GTR contract. In the case of skin-painting studies where large amounts of con-" * .~ densate are required, this will have to be purchased by CTR at the cost incurred ~" A: by necessary addition of technical personnel for the production of these large ~~ '`amounts of condensate. However, it is anticipated' that the new condensate ma-~ -: ~10 Additional Information (Including relation of work to other projects and other sources of support) chine will be so ~~ -~i.r .. . .:. . . .. . . - .,,. ,.,'•. the past, it has been possible to share some personnel with much more. efficien ' ~` . W , r 'carcinogenesis studies carried out under National - Institutes than existing:, s_ ~,mok ~ri ou 'ng ~of Health, National Cancer Institute Research Grant No. CA machines that this cos , ~~04869. Since this grant has been discontinued, this is no longer will be small. ~~ possible. :.Thus,` support for these studies by CTR is assuming added importance.~ ' for us. `°Even with it (at the requested rate), we must reduce our total effort iri the carcinogenesis field. Without CTR support, we should have drastically to ,.~reduce our efforts in this field in which we have worked since 1948 and for which 34, 760. 5,300, 3,085_ 6, 000_ 6,472, Total 55, 617. the Insti)ution ' . ~3. aYr irl.+T l ® 0 Y N m we have developed a uniquely competent team. Signature Director of Project ll, .~ ^ Business Officer of .w