Comparison between in vitro and in vivo tests for carcinogenicity

205

Mutation Research,

75 (1980) 205--213 © Elsevier/North-Holland Biomedical Press

ICPEMC WORKING PAPER 2/1 COMPARISON BETWEEN IN V I T R O AND IN VIVO TESTS F O R CARCINOGENICITY AN O V E R V I E W *

DAVID B. CLAYSON Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105 (U.S.A.)

(Received 24 August 1979) (Accepted 7 September 1979) Summary There are examples of short-term prescreening tests for carcinogenicity that fail to agree with the results o f animal bioassays. Factors which m a y lead to such discordant results are discussed in terms of the present understanding of the mechanisms of chemical carcinogenesis and the quality of the results obtained in vivo and in vitro. 1. Need for economical cancer tests The recognition that certain man-made and naturally occurring chemicals induce cancer, combined with the present high level of the human cancer burden, emphasizes the need for identification of chemical carcinogens. For the past 40 years, the administration of chemicals to animals has been the only reliable m e t h o d for the experimental identification of carcinogens [6,18]. Such T h i s p a p e r is b a s e d o n a p r e s e n t a t i o n g i v e n a t t h e f i r s t m e e t i n g o f C o m m i t t e e 2 o f t h e I n t e r n a t i o n a l Commission for Protection against Environmental Mutagens and Carcinogens (ICPEMC), Versailles, France, December 4--7, 1978. It h a s b e e n a ~ r e e d t o p u b l i s h t h i s d o c u m e n t as a w o r k i n g p a p e r f o r C o m m i t t e e 2 o f I C P E M C . T h e v i e w s e x p r e s s e d are t h o s e o f t h e a u t h o r a n d d o n o t n c c e s s a l d l y r e p r e s e n t t h o s e o f t h e C o m m i s i o n . T h e y a r e p r e s e n t e d t o s t i m u l a t e d i s c u s s / o n a n d c o m m e n t s will b e w e l c o m e d b y t h e a u t h o r . I C P E M C is a f f i l i a t e d w i t h t h e I n t e r n a t i o n a l A s s o c i a t i o n o f E n v i r o n m e n t a l M u t a g e n S o c i e t i e s a n d is s p o n s o r e d b y t h e I n s t i t u t d e la Vie. All c o r r e s p o n d e n c e w i t h I C P E M C s h o u l d b e a d d r e s s e d t o t h e s e c r e t e r y : Dr. P . H . M . L o h m a n , M e d i c a l B i o l o g i c a l L a b o r a t o r y T N O , P . O . B o x 4 5 , 2 2 8 0 A A Rijsw i j k ( T h e N e t h e r l a n d s ) . Tel: 1 5 - 1 3 8 7 7 7 , t e l e x 3 1 1 0 1 p m t n o n l . (ICPEMC document: 78-1979-WP2/1-C2)

206 tests, if done adequately, are expensive. For example, a relatively simple test carried o u t at t w o dose levels in two species [24] n o w costs a b o u t U.S. $ 500 000. The facilities for conducting such tests are limited, both by the absence of sufficient, trained personnel and by the lack of well
development Theoretically, at least, if one could identify one or more critical stages in cancer induction, it should be possible to devise relatively accurate short-term tests for carcinogenic chemicals. Thus, for example, if the interaction of a chemical carcinogen with DNA were a critical stage in chemical carcinogenesis, one should be able to devise a rapid test for chemical carcinogens based on this observation. We know enough a b o u t the probable mechanism of chemically induced cancer today, at least to understand w h y our present battery of tests is, at worst, moderately successful. Chemical carcinogenesis m a y be divided into three broad, possibly overlapping, phases: (A) The carcinogen either decomposes spontaneously or is converted through one or more stages enzymaticaUy to a highly reactive, positively charged entity called an electrophile [18,19]. Thus, aromatic hydrocarbons, such as b e n z o [ a ] p y r e n e lead to bay region diol-epoxides, aromatic amines to derivatives of related arylhydroxylamines, and nitrosamines to their a-hydroxylated form. Each of these intermediates is then able to decompose spontaneously to a highly reactive electrophile. The reactivity of these electrophiles may well have to lie within critical limits if they are to react with their critical target. Some electrophiles may be so reactive that they are n o t sufficiently stable to reach their critical target, others m a y be so stable that under the conditions normally pertaining in the b o d y , t h e y fail to react to a significant

207 extent, although they m a y do so in vitro. This hypothesis has not, as yet, been experimentally tested. (B) The second stage of the carcinogenic process is the interaction of the electrophiles with any electron-rich groups in the surrounding tissues. Such electron-rich groups occur in cellular DNA, R N A and protein, as well as small molecules. The critical target must be presumed to be among the nucleophiles undergoing such an interaction, b u t its exact nature is n o t known. Present evidence indicates that the critical cancer induction targets are probably genetic, that interaction with DNA is critical, and that cancer is a genetic t y p e event. Evidence can, however, be adduced that epigenetic processes are involved. Two events may follow carcinogen interaction, b o t h affecting the cell genome. One is the influence of cell replication which may "lock in" the damage induced into DNA by carcinogen interaction. The alternative is the induction of prereplicative DNA repair which may be a restorative process or m a y also contribute to the "locking in" of errors. Among those cells whose genome is modified b y carcinogen interaction must be tumor-progenitor cells which, if they can multiply, lead to frank clinical tumors. (C) The third stage of t u m o r induction is the development of the tumor-progenitor cells to frank neoplasia. This is the least understood part of the whole carcinogenic process. Progenitor cells must have some proliferative advantage over their normal counterparts. The nature of the humoral or local factors which mediate this action is n o t known, b u t probably includes cell proliferation, hormonal and chalone influences, immunologic processes and many others. Each of these stages of carcinogenesis may be modified b y a variety of agents. For example, the a m o u n t of procarcinogen converted to an electrophile is altered b y enzyme-inducing agents, the site and magnitude of DNA--electrophile interaction is d e p e n d e n t on cell proliferation, while t u m o r development is under the control of many factors, including the immune system, hormones, retinoids, promoting agents, and so forth. Experience in the use of short-term tests to monitor such modifying agents is being developed. If a short-term test is to be a truly accurate predictor of carcinogenicity, it should match each of the critical events in carcinogenicity. Currently available tests, as discussed below, generally are models of only one of these events. 3. Metabolism in vivo and in vitro The metabolic activation of a carcinogen is a critical event in the carcinogenic process. It does n o t lend itself to the development of short-term tests because the electrophilic intermediates or their immediate precursors are, as far as is known, usually t o o unstable to be readily isolated and characterized or identified. Metabolism in vivo and in vitro will n o t be discussed in detail. There are, however, certain specific differences between in vivo and in vitro metabolism which may be of considerable importance to the accuracy of short-term test results. The microsomal, or $9 fraction, normally used in the Ames test, etc., undergoes some pretty rough treatment during its preparation. Thus, it is possible that activating and deactivating enzymes may be dissociated from each

208 other. Garner et al. [11], for example, found rat-liver $9 fraction was able to activate aflatoxin B, (AFB1) t o a bacterial mutagen. This is in good agreement with results f o u n d in vivo. In the hamster, which responds only poorly if at all to AFB~ in vivo, the $9 liver fraction provided a much superior activating system, even to that of the rat-liver $9 fraction. This effect m a y be due to the fact that during preparation of the $9 fraction, activating and deactivating enzymes are dissociated. A further possible difficulty in using the $9 fraction is that, despite addition of cofactors, their concentrations may n o t be the same as those that occur in vivo. For these reasons, the upcoming generation of mammalian~ell mutation tests in which cells capable of metabolic activation are incubated in the presence of mutable cells m a y more closely resemble metabolic activation and deactivation that occurs in vivo [14,15]. There is one further area o f difficulty which m a y lead to confusion in metabolic activation. In vivo systems are protected b y urinary, biliary and respiratory excretion systems. In vitro, these systems do n o t exist and the carcinogencontaining medium remains in contact with the mutable cells for prolonged periods. In consequence, less reactive metabolites may persist for considerable periods in the in vitro medium and possibly lead to positive results n o t found in vivo. Specific examples of this p h e n o m e n o n are not easy to d o c u m e n t at this time, and must be regarded as speculative. The demonstration by Raha et al. [22] that the K-region epoxide of b e n z o [ a ] p y r e n e interacts in vitro with protein provides evidence for the effect discussed. 4. Interactions between carcinogens and tissue macromolecules The binding of carcinogens to tissue macromolecules has has been extensively studied, particularly binding to DNA, R N A and protein. Major emphasis has been placed on binding to DNA, largely because the macromolecule is, as the genetic material of the cell, to some extent irreplaceable, and thus, indicates that a single lesion might have some effect on the cell's progeny, such as demonstrable mutation. DNA or other forms of binding do not, however, appear to correlate well to carcinogenicity of individual chemicals. This is possibly because n o t all forms of binding to DNA have an equivalent effect. It is well-known, for example, that while dimethylnitrosamine is a p o t e n t rodentliver carcinogen even in single doses [16], in the rat methylmethanesulfonate is ineffective in liver [5]. Dimethylnitrosamine methylates the 0-6 and No7 positions of guanine, whereas methyl methanesulfonate alkylates mainly the N-7 position [21]. The significance of this to the cell genome was demonstrated b y Ludlum and his colleagues [12], who showed that while N-7 methylpolyguanosine replicated faithfully, 0 - 6 methylpolyguanosine replicated unfaithfully. This, to some extent, may explain the relative effectiveness of the t w o chemicals in inducing liver cancer. One other form of DNA--chemical interaction exhibits an effect w i t h o u t covalent binding. This is intercalation of chemicals between the DNA bases, which leads to frameshift mutations in some bacteria, b u t does n o t seem wellcorrelated with carcinogenicity, unless covalent interaction accompanies intercalation. Thus, acridines, such as acridine orange, do n o t appear on present evidence to be p o t e n t carcinogens unless t h e y are metabolized in the rodent

209 liver [7]. Acridine orange, for example, is a potent intercalator, but a relatively weak rodent-liver carcinogen. Hycanthone also intercalates. It is a potent microbial mutagen, but at most, a borderline mouse-liver carcinogen [4]. The recent reports that certain naturally-occurring anthraquinones are frameshift mutagens in S. typhimurium TA1537, but not in other tester strains [3], may also be due to their ability to intercalate with DNA. Aminoalkylaminoanthraquinones interact with DNA probably by intercalation [10], but neither intercalation nor carcinogenicity has been studied with these naturally-occurring derivatives. Our present battery of short-term prescreening tests depends largely on the consequences of covalent interaction between chemical carcinogens and DNA and consists of tests depending on microbial or mammalian cell mutation, DNA repair and cell transformation. The latter alone may depend on factors other than DNA interaction. This evidence does not prove carcinogenesis is a mutational process, it only makes it likely that some interaction of the electrophile with a critical cellular receptor is necessary. Nevertheless, the apparent parallelism between mutational events in micro-organisms and mammalian cells and carcinogenesis in animals presently is rather impressive. 5. Critical events in the development of chemical neoplasia In my view, there are no presently known critical events in tumor development, as opposed to initiation. A number of tissue-specific changes have, from time to time, been described, such as hyperplasiagenesis to the urinary bladder [9], or the sebaceous gland suppression test in mouse skin [13], but these do not qualify in terms of general application nor specificity [8]. The chemical induction of cancer in animals involves each of these 3 stages (metabolic activation, interaction and tumor development) outlined above. Difficulties arise in the extrapolation of carcinogenicity data obtained in animals to man because of species differences in pharmacokinetic distribution of the test agent, in the levels of act'ivafing and detoxifying metabolic enzymes, and possibly in the level of noncritical receptors that may interact with the electrophile and thus serve to protect the critical receptor(s) from chemical carcinogens. Both qualitative and quantitative differences in factors affecting tumor development have been recognized. For example, female rodents exhibit an estrous cycle, women, a menstrual cycle. 6. Assessment of the quality of in vitro and in vivo evidence

(A) In vivo. There is little difficulty in accepting as carcinogens chemicals which have repeatedly led to high yields of tumors in experiments involving several animal species. Chemicals, such as 7,12-dimethylbenz[a]anthracene, 4~aminobiphenyl or diethylnitrosamine, are undoubtedly potent carcinogens on any criteria. More difficulty arises with agents which induce limited tumor responses in a single species and/or single testing protocol. Saccharin, which appears reproducibly only to induce bladder carcinoma in rats treated from conception to death, is an example of a very limited form of carcinogenicity Other examples of the difficulty of deciding whether a chemical is carcinogenic

210 are f o u n d with chemicals tested in too few animals or with which survival was inadequate [6]. The j u d g m e n t as to whether a chemical is not carcinogenic is much more difficult. It is clearly necessary to determine the minimum evidence on which a chemical can be accepted as noncarcinogenic. One possibility is that to be accepted as noncarcinogenic, a chemical should fail to induce tumors in a wellconducted study carried o u t under the U.S. National Cancer Institute Bioassay Protocol [24] or its equivalent in other bioassays. Such a protocol involves two species, two dose levels (excluding controls), both sexes and 50 animals in each subgroup. The so-called " m a x i m u m tolerated d o s e " is mandated in this assay. If a short-term in vitro test shows t h a t a chemical is positive, despite such a level of negative in vivo evidence, there is cause for concern. In the case of less adequate or deficient negative tests, it is possible t h a t the in vivo rather than the in vitro system is discordant. (B) In vitro. There is little d o u b t of the significance of in vitro tests in which the test agent induces an effect which is m a n y f o l d higher than t h a t seen in appropriate controls. The smaller effects, particularly those induced by high concentrations of test agents, need more careful consideration. Workers in the short-term area are beco_ming more aware of the need for statistical analysis in validation of their results, but in m y opinion, much thought must be given to the m e t h o d of analysis as shown by the following questions: (1) Should analysis be based on the number of plates examined or the number of cells? (2) Should the number of m u t a n t s (transformants) be based on the actual n u m b e r observed or be corrected for cell survival? Put another way, if control plates show two mutants/106 cells and test plates two mutants/10 s surviving cells, should we assume that if all 106 cells had survived, there would be 20 mutants on the test plate, i.e., t h a t chemical toxicity has affected only those cells which do n o t normally give rise to mutants? In other words, does the agent induce or select for mutation? (3) Should only c o n c o m i t a n t controls be used for statistical analysis, or if historical controls show gross variation, should results t h a t do n o t statistically exceed the highest historical control be discounted? Each of these questions is scientifically answerable, but may require detailed investigation of individual chemicals. 7. Correlation between in vivo and in vitro results It has been suggested, for example, t h a t the Ames S. typhimurium microsome test agrees 80--90% with long-term bioassay results [1]. The reason for the discrepancy has n o t y e t been actively pursued, despite the fact t h a t some t h o u g h t might help in understanding the usefulness of tests as predictors of carcinogenicity. Clearly, inadequate in vivo data is one reason for lack of agreem e n t between the long-term and rapid tests. A second factor may be that specific in vitro tests m a y be more adequate for some classes of chemicals than others. The Ames test, until the protocol was modified by exposure by liquid preincubation, was inadequate, to say the least, for nitrosamines. It is still inade-

211 quate for chlorocarbon carcinogens, hydrazines, estrogens, and possibly, for metals. There is a need to k n o w the chemical--structural specificity for each short-term test. Despite such considerations, there is still another possibility. We have already identified chemicals, which although positive in animal experiments, are negative or just borderline in our current battery of short-term tests, as, for example, diethylstilbestrol, saccharin and nitrilotriacetic acid. In m y view, such substances give us difficulty because present definitions of a carcinogen are inadequate insofar as mechanisms of action are concerned [6,25]. Presently, any agent which significantly increases the incidence of malignancies in a population is regarded as a carcinogen, no matter what the mechanism. Thus, in addition to the classical or " g e n o t o x i c " carcinogens, there m a y be others which enhance t u m o r yield by some other mechanism. For example, trichloropropene oxide inhibits epoxide hydratase, which may explain w h y Berry et al. [2] showed it enhanced 3-methylcholanthrene carcinogenicity to mouse skin. Other agents may increase the cell proliferation rate in liver and enhance t u m o r yield in this way. The fact that the in vivo demonstration of carcinogenicity is n o t confirmed b y in vitro genotoxic tests does not necessarily indicate that the tests are deficient. It may indicate that certain agents induce tumors by non-genotoxic mechanisms. 8. Research-oriented use of in vitro tests Short-term tests should n o t only be useful for the possible replacement of chronic animal bioassays, b u t m a y be of major value for the advancement of knowledge. For example, it is clear that liver is n o t the only tissue capable of metabolically activating chemical carcinogens. McLean and Magee [17] showed that a zero protein diet inhibits the capacity of the liver to metabolize dimethylnitrosamine, b u t had no effect on the level of metabolism in the kidney. Short-term tests are potentially useful because they amplify the metabolic activation of carcinogens by less metabolically active tissues. My colleague, Dr Langenbach and his associates, have recently shown that mammalian cell mutation o f V79 cells, primary explants of bladder, lung and liver cells activated carcinogens. With the Ames Test, they found that $9 from bladder is effective for metabolic activation. My colleague, Dr. Mirvish, using inhibition of tritiated thymidine incorporation into DNA, has presented indirect evidence that esophageal epithelium activates specific nitrosamines [20]. Short-term and other tests offer an o p p o r t u n i t y to define the metabolic activating capacity of specific tissues and ultimately, of human tissues, which are usually so difficult to obtain in quantity sufficient for classical metabolism studies. Short-term tests could be used to demonstrate the capability of human tissues to metabolically activate specific carcinogens. This could be a most useful approach to the relevance of specific animal carcinogens to t u m o r formation in man. 9. Risk assessment The suggestion that particular chemicals are carcinogenic to animals is only half the battle in meaningful cancer prevention. As, or more, important is the risk they present to individual men and w o m e n in the c o m m u n i t y . At this time,

212 m o s t workers see little, if any, correlation between potency in short-term tests and carcinogenic potency in animals. Ames [1] discussed a very limited range of chemicals using the S. typhimurium test. The qualitative correlation of carcinogenic potency in animals and that seen in the few examples adequately studied in man is likewise open to serious questions. 10. Conclusions There can be little d o u b t that in the near future, the value of short-term tests will be recognized by both scientists and regulators. While these tests presently offer great promise in recognizing possible environmental carcinogens in an economic manner, some of the difficulties alluded to in this presentation make it clear that much more research and development is needed before they can be accepted as alternates to long-term chronic carcinogenicity bioassays in small rodents. The development o f families of short-term tests is advocated as the most promising way to ensure that potent carcinogens with a high human risk potential do not escape the short-term carcinogen-screening tests. Such families o f tests must, however, be assembled in the light of firm knowledge of the capabilities and tendency toward error of each test included. References 1 Ames, B.N., E n v i r o n m e n t a l chemicals causing cancer and genetic bi rt h defects: developing a strategy for min imizin g h u m a n exposure, Calif. Policy Seminar, Dec. 14, 1977, pp. 1--37. 2 Berry, D.L., T.S. Slaga, A. Viaje, N.M. Wilson, J. Dlgiovanni, M.R. J uc ha u and J.K. Selkirk~ Effect of tr iehloro pene oxide on the ability of p o l y a r o m a t i c h y d r o c a r b o n s and their "K-region" oxides t o initiate skin t u m o r s in mice and to bind to DNA in vitro, J. Natl. Cancer Inst., 58 (1977) 1051--1055. 3 Brown, J.R., and P.S. Dietrich, Mutagenicity of a n t h r a q n i n o n e and b e n z a n t h r e n e derivatives in the S a l m o n e l l a / m i c r o s o m e test: activation of a n t h r a q u i n o n e glycosides by enzymic e xt ra c t s of rat cecal bacteria, Mutation Res., 66 (1979) 9--24. 4 Bulay, O., H. Urman, K. Patti, D.B. Clayson and P. Shubik, Carcinogenic p o t e n t i a l of h y c a n t h o n e in mice and hamsters, Int. J. Cancer, 23 (1979) 97--104. 5 Clapp, N.K., Carcinogenicity of nitrosamines and m e t h a n e s u l p h o n a t e esters given i nt ra pe ri t one a l l y in R F mice, Int. J. Cancer, 12 (1973) 728. 6 Clayson, D.B., Chemical Carcinogenesis, Churchill, London , 1962. 7 Clayson, D.B., Carcinogenic and anti-carcinogenic properties of acridines, in: R.M. Acheson (Ed.), The Acridines, 2rid edn., Interscience, New York, 1973. 8 Clayson, D.B., Historical evohition of short-term tests for carcinogenicity, in: L. Golberg (Ed.), Carcinogenesis Testing of Chemicals, CRC Press, Cleveland, Ohio, 1974, p. 79. 9 Clayson, D.B., T.A. Lawson, S. Santana and G.M. Bonser, Chemical i n d u c t i o n of hyperplasia and of malignancy in the mouse bladder epithelium, Br. J. Cancer, 19 (1965) 297. 10 Double, J.C., and J.R. Brown, The i n t e r a c t i o n of a m i n o a l k y l a m i n o a n t h r a q u i n o n e s with deoxyribonucleic acid, J. Pharm. Pharmacol., 7 (1975) 502--507. 11 Garner, R.C., E.C. Miller and J.A. Miller, Liver microsom a l m e t a b o l i s m of aflatoxin B 1 to a reactive derivative t o x i c to Salmonella typhirnurtum TA1530, Cancer Res., 32 (1972) 2058--2066. 12 Gerchmann, L.L., and D.B. Ludhim, The properties of O6-methylguaninc in t e m p l a t e s for RNA polymerase. Proc. Am. Assoc. Cancer Res., 14 (1973) 13. 13 Healey, P., L.E. Mawdesley-Thomas and D.H. Barry, The effect of some polycyclic h y d r o c a r b o n s and tobacco condensates on non-specific esterase activity in sebaceous glands of mouse skin, J. Pathol., 105 (1971) 147. 14 Huberman, E., Correlation b e t w e e n m u t a g e n i c i t y and carcinogenicity, Mammalian cell t r a n s f o r m a t i o n and cell m e d i a t e d mutagenesis by carcinogenic polycyclic hydroc a rbons , Mutation Res., 29 (1975) 285.

15 Langenbach, R., H.J. Freed and E. Huberman, Liver cell-mediated mutagenesis of m a m m a l i a n cells by liver carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 75 (1978) 2864. 16 Magee, P.N., and J.M. Barnes, Carcinogenic nitroso c o m p o u n d s , Adv. Cancer Res., 10 (1967) 163. 17 McLean, A.E,M., and P.N. Magee, Increased renal carcinogenesis by d i m e t h y l n i t r o s a m i n e in proteindeficient rats, Br. J. Exp. Pathol., 51 (1970) 587.

213 1 8 Miller, E . C . , a n d J . A . Miller, C h a p t e r 1 6 : T h e m e t a b o l i s m o f c h e m i c a l c a r c i n o g e n s t o r e a c t i v e e l e c t r o p h i l e s a n d t h e i r p o s s i b l e m e c h a n i s m s o f a c t i o n i n c a r c i n o g e n e s i s , in: C . E . Searle ( E d . ) , C h e m i c a l Carcinogens, New York, A.C.S. Monogr. No. 173, 1976, p. 737. 1 9 Miller, J . A . , C h e m i c a l c a r c i n o g e n e s i s - - a n o v e r v i e w . G . H . A . C l o w e s M e m o r i a l L e c t u r e , C a n c e r R e s . , 20 (1970) 559. 2 0 Mirvish, S.S., C. C h u a n d D . B . C l a y s o n , I n h i b i t i o n o f [ 3 H ] t h y m i d l n e i n c o r p o r a t i o n i n t o D N A o f r a t esophageal epithelium and related tissues by carcinogenic N-nitroso compounds, Cancer Res., 38 (1977) 458. 21 O ' C o n n o r , P . J . , M . J . C a p p s , A.W. C r a i g , P . D . L a w l e y a n d S.A. S h a h , D i f f e r e n c e s in t h e p a t t e r n s o f m e t h y l a t i o n in r a t liver r i b o s o m a l r i b o n u c l e i c a c i d a f t e r r e a c t i o n i n vivo w i t h m e t h y l m e t h a n e s u l p h o n a t e a n d N , N - d i m e t h y i n i t r o s a m i n e , B i o c h e m . J., 1 2 9 ( 1 9 7 2 ) 5 1 9 . 2 2 R a h a , C., C . H . G a l l a g h e r , P. S h u b i k a n d S. P e r a t t , C o v a l e n t b i n d i n g t o p r o t e i n o f t h e " K - r e g i o n " o x i d e o f b e n z o [ a ] p y r e n e f o r m e d b y m i c r o s o m e i n c u b a t i o n , J. N a t l . C a n c e r I n s t . , 5 7 ( 1 9 7 6 ) 3 3 . 2 3 S m i t h , W . E . , S e b a c e o u s g l a n d a c t i v i t y as a m e a s u r e o f t h e r e l a t i v e c a r c i n o g e n i c i t y o f c e r t a i n oils a n d t a r s , Bull. N . Y . A c a d , M e d . , 3 2 ( 1 9 5 6 ) 7 7 . 2 4 S o n t a g , J . M . , N . P . P a g e a n d U. S a f f l o t t i , G u i d e l i n e s f o r C a r c i n o g e n B i o a s s a y i n S m a l l R o d e n t s , N C I C G o T R - 1 , U . S . D e p t . H l t h . , E d u c . a n d W e l f a r e , P u b l . H l t h , Sci., W a s h i n g t o n , D . C . , 1 9 7 6 . 25 Subcommittee on Environmental Carcinogenesis, National Cancer Advisory Board, National Cancer Inst. General Criteria for Assessing the Evidence for Carcinogenicity of Chemical Substances: Report of the Subcommittee on Environmental Carcinogenesis, National Cancer Advisory Board, J. Natl. Cancer Inst., 58 (1977) 461---463. 26 Wright, A.S. ICPEMC working paper 2/2, The role of metabolism in chemical mutagenesis and chemical-carcinogenesis, Mutation Res., 75 (1980) 215--241.