Chem. Res. Toxicol. 1992,5, 737-741
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Forum Dose Selection for Animal Carcinogenicity Studies: A Practitioner’s Perspective Richard A. Griesemert National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709 Received September 14, 1992
Conceptual approaches to the design of experiments, including the selection of doses, are part of the training of every toxicologist. Occasionally, a question is raised about the appropriateness of dose selection in a particular experiment, but as the principles of dose selection have not changed much over time, the dose selection process itself has rarely been an issue. What has come into question recently, however, is the relevance of so-called high-dose animal carcinogenicity studies for humans. I present here the basis for top dose-setting in carcinogenicity studies. From the viewpoint of someone who has been engaged in such studies, I have attempted to temper expectations about trans-species concordanceand to convince the reader that the present methods for selecting top doses are not only desirable but necessary. For a given substance the optimally effective high dose establishes a point of departure for all the other experimental studies to follow.
whether or not the substance has carcinogenic activity and, if so, the types of tumors produced and the parts of the body affected.
Not Too High, Not Too Low, but Just Right
Y the MTD
Selectingthe top dose level is one of the most important design considerations for animal carcinogenicity studies (I). If the dose level selected is too high and produces toxic effects, decreased longevity or inhibited growth of the animals might interfere with the expressionof potential carcinogenicity. Dose levels that are too low might not produce effects that can be detected in small groups of animals. Somewhere between is an optimal dose level for detecting and measuring carcinogenic effects. When the study is designed, the most effective dose level is not yet known. That is one of the objectives of the study. The adequacy of the dose levels selected can only be assessed retrospectively after the study has been completed.
If animals were to be used as surrogates for humans to detect carcinogenic effects in say one in a million individuals at some level of exposure, and if it is assumed that mice and rats are similar in susceptibility to humans, then the experiment would require a million rats or mice. As that is clearly not practicable, smaller groups of animala are typically employed (on the order of 50 or 100) but exposed at higher dose levels. It is expected that with increasingly higher doses the tumor response will be proportionately greater. For the purpose of finding whether or not a substance has carcinogenic activity, the dose level has to be high enough to produce a measurable effect (about a 10% or greater excess tumor response of a particular type and site in a group of 50 animals, depending on a great many considerations including the background rates of spontaneous tumors). There is a limit, however, to the amounts that can be administered to animals because both carcinogenic and noncarcinogenicsubstanceshave toxic effectsat high doses. It is necessary therefore to evaluate noncancer toxicity for each substance before cancer studies can begin. Typically, a series of toxicity studies are conducted in which animals are exposed to a range of dose levels for as many weeks or months as it takes to learn the nature of the toxic effecta and the dose levels that produce them. On the basis of the data obtained, including histopathologicalevaluations of the lesions produced, an estimate is made of the highest
Typecasting Animal carcinogenicity studies may be of several types. Those of concern have are intended primarily for the identificationof carcinogenichazards. Subsequent studies may be designed to provide information about doseresponse relationships, mechanisms of action, chemical interactions, or chemoprevention and chemotherapy. Animal studies are sometimes designed to fulfill multiple purposes simultaneously, but more definitive and less expensive studies can be designed once it is determined ~~
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Deputy Director, NIEHS.
The Grand Design Animal carcinogenicity studies are based on two fundamental principles of toxicology: (a) that effectsproduced by a substance in laboratory animals are applicable to humans and (b) that exposure of experimental animals to toxic agents in high doses is a necessary and valid method of discovering possible hazards in humans. In a typical carcinogenicitystudy, the suspect material is administered to groups of animals (both sexes of two species) for the majority of their lives and the resultant tumor patterns are compared with those in control animals not administered the test substance. Details of the experimental designsand statistical considerations can be found in IARC Scientific Publications (2, 3).
This article not subject to U.S.Copyright. Published 1992 by the American Chemical Society
738 Chem. Res. Toxicol., Vol. 5, No. 6,1992 Table I. Comparison of Carcinogenic Doses to Humans and Rodents for Selected Substances
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~
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typical doses associated with cancer in humans
human carcinogen
carcinogenic doses to animals
chlorambucil
0.1-0.2 mg/kg PO or 10-20 mg PO daily in 2-week courses cyclophosphamide 10-15 mg/kg iv, 3x/wk, or 1-5 mg/kg PO diethylstilbestrol 1mg PO daily during pregnancy (18 pg/kg/day) melphalan 2-6 mg PO daily for 15-102 months (total 13-260 mg/kg) analgesics containing phenacetin phenacetin
> 1 g/day for >1yr (1.5-27 kg)
PUVA (8-methoxypsoralen and UV)
20 mg PO daily or 1% topical 8-methoxypsoralen plus UV
1.5-4.5 mg/kg ip, 3x/wk 0.63 mg/kg, 5x/wk, drinking water 6 pg/kg/day PO 0.75-1.8/mg/kg ip, 3x/wk for 6 months (total 59-140 mglkg)
1.25% or 2.5% in diet for 18 months 0.2% in diet 5-250 pg topical daily plus UV 0.04 mg ip
Table 11. Examples of Recent NTP Long-Term Studies Performed at Dose Levels to Which People Are Exposed TLV
substanceo tetranitromethane
1ppm
1,3-butadiene ozone
PPm EPA standard 0.12 ppm
NTP rat 0 , 2 , 5 ppm mouse 0,0.5, 2 ppm mouse 0,625,1250 ppm rat and mouse 0,0.12, 0.5,l.O ppm
0 Tetranitromethane and 1,3-butadiene were both carcinogenic to animals a t the doses tested. The ozone studies are still in progress.
Griesemer
should not expect exact concordance between results in experimental animals and humans even at the same dose levels. People are heterogeneous in respect to genetic constitution, diet, lifestyle, etc., and are therefore not exactly comparable to the groups of laboratory animals that are selected to be homogeneous in virtually all respects except for exposure to the test substance. Moreover, the patterns of exposure of humans are not likelyto be regular and repetitious as for the animals but intermittent and variable, extending oftentimes over several decades. It has been commonly observed, too, that small animals, with rapid metabolism, sometimes require larger doses of a drug to achieve the same pharmacologic effect as much smaller doses per unit weight for larger animals and humans. The evidence a t hand indicates that, in general, carcinogenicity studies in rodents appear to be very good qualitative predictors of carcinogenicity for other mammalian species. Their value as quantitative predictors is also quite good in those instances where comparative species’ pharmacokinetic and toxicokinetic data are available. A comparison can be made for those substances where data exist on carcinogenic doses to both humans and animals (Table I). For these substances the carcinogenic doses in man and animals are about the same order of magnitude. It should be emphasized, too, that NTP animal studies often include dose levels to which some people have been or are being exposed (Table 11).It is not practicable, of course, to test all the dose levels to which all people may be exposed to every substance, but the “high doses” used in animal carcinogenicity studies cannot be considered unrealistic for those humans in the high exposure ranges.
The Good News
dose level that can be administered wihout interfering significantly with the growth, well-being,and longevity of the animals, except for the development of cancer. The dose estimates take into account biologic responses such as the adaptation of the tissues to repeated exposures and the cumulative toxicity produced by certain substances. Thus, the high dose tested in carcinogenicity studies, sometimes referred to as the maximum tolerated dose (MTD)’ or the estimated maximum tolerated dose (EMTD), is not so high as to be detectably toxic but is set just at the margin of the range of toxicity (too low a dose level to serve by itself as a study of noncancer toxicity). Because of the uncertainty of accurately predicting the MTD, a second lower dose level (some fraction of the EMTD) is usually used in the event that the high dose turns out to have been too high. Moreover, when studies at multiple dose levels reveal dose-related trends in tumor response, there is greater confidence that the observed effects are related causally to administration of the test substance.
Great Expectations Carcinogenicity studies in animals are powerful tools. They provide biologic plausibility for the epidemiologic associations between exposure to a substance and cancer formation. Animal studies also provide evidence for causality because they are conducted under laboratory conditions where the variables can be controlled. But one ~
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Abbreviations: MTD, maximum tolerated dose; EMTD, estimated maximum tolerated dose; NTP, National Toxicology Program; TCDD, tetrachlorodibenzodioxin;NCI, National Cancer Institute. 1
Carcinogenicity studies in animals at high doses Definitively identity substances with carcinogenic activity for animals. Provide information about relative potency. Characterize the tumor response as to tumor types, sites of origin, and malignant potential. Provide information about generalizability among sexes and species. Provide clues to mechanisms of action. Permit cross-comparisons, including structureactivity relationships, that improve predictive capabilities. Provide information about the lack of carcinogenic activity for many substances and thus direct public health attention toward more important problems.
The Not So Good News Carcinogenicity studies in animals at high doses 0
Provide no direct information about effects a t dose levels lower than those used in the studies. Sometimes produce equivocal or marginal results that require replication or other additional studies. Provide limited information about mechanisms of carcinogenicity (although they do assist greatly in predicting what mechanistic studies might profitably be considered).
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Do not by themselves provide information about exposure scenarios other than those studied.
Too Many Carcinogens? The utility of animal carcinogenicity studies depends partly on the true proportion of environmental chemicals that have carcinogenic activity. If all or nearly all substances are carcinogenic under some circumstances, qualitative screens in animals are not needed. If relatively few substances are carcinogenic, special attention should be given to ensuring that the methods used reliably detect them. The data at hand, unfortunately, are too limited to provide much insight into this question. Fewer than 1000 chemical substances have been adequately tested in both sexes of two rodent species and reported in the open literature. The largest single data base comes from U S . National Toxicology Program (NTP) studies that used sufficiently standardized methods to permit cross-comparisons. To date, NTP studies have been completed on 430 chemical substances, and of these about one-fourth gave strong evidence of carcinogenic activity (usually in both species) and another fourth gave evidence that was limited to weak or marginal responses at one site or in one sex of one species. The remaining half of the substances tested lacked carcinogenic activity under the conditions of the tests. Does this mean that a fourth or half of the more than 10 OOO OOO chemical substances are carcinogens? The answer is no because the substances tested were not randomly selected. The NTP has deliberately selected substances for study that had a suspicion of carcinogenicity on the basis of prior published toxicity data and/or on the basis of structural resemblance to known carcinogens. There has been little change in the selection criteria between the 1970s and 1980s (Table 111). Limited resources have not been spent on substances considered not likely to be carcinogenic. A more answerablequestion than how many carcinogens there might be is whether subclasses or groupings of chemicalsubstances can be formed in which the members are either carcinogens or noncarcinogens. For example, NTP has studied eight soluble benzidine dyes, each of which produced a tumor pattern consistent with that produced by benzidine itself. On the other hand, an insoluble benzidine-containing pigment (diarylanilide yellow) lacked carcinogenic activity. I suggest we may want to know something about the bioavailability of benzidine from the other 200 benzidine-based dyes but that long-term studies should not be given a high priority. Claims that there are too many carcinogens should be considered as value judgments rather than scientific judgments; however many there may be is what there are.
Toxicity Causes Cancer? Experimental attempts to dissociate noncancer toxicity and carcinogenicity have been going on for at least 50 years. The underlying problem historically has been that limiting dilution (lower dose) experiments in small groups of animals have usually lacked the statistical power to detect possible carcinogenic effects. Today we know how to design the necessarily unbalanced experiments, but at dilutions below MTD/10 or so the numbers of animals
Table 111. Comparison of Recent and Previous Deliberate Bias in Selecting Substances for NTP Long-Term Animal Studies 1985-1990,59Substances (A)37 (63% ) based primarily on exposure structural relationship to a known carcinogen (IO)or class of carcinogens (20) suspicion of carcinogenicity based on short-term study results (7) (B)16 (27% ) exposure gap in knowledge about an entire chemical class
(C)4 (7%) exposure, primarily
(D)2 (3%) gap in knowledge
1975-1978,104 Substances (A) exposure and suspicion of carcinogenicity, 65 (63% ) exposure data from previous studies structural relationship to a known carcinogen member of a chemical class containing carcinogenic chemicals potential for metabolism to a carcinogenic intermediate (B)exposure and a gap in knowledge, 39 (37%)
needed to retain statistical power generally become prohibitively expensive. My colleagues and I, using classical clinical and pathological methods to evaluate toxic effects, have found almost no correlations between the types and sites of early or late toxic effects and cancer formation in animal studies (4,5). The possibilities still exist that finer measures of toxicity a t the cellular and molecular levels or measures of physiologic perturbations might correlate with cancer formation. The problem here is to demonstrate causal associations between (a) early occurring, frequent, widespread effects (adduct formation, altered oncogene expression, increased cell division, etc.) and (b) late developing, rare, focal effects (cancer). Research in this field is active and better information is anticipated, especially from investigators working with groups of substances that have similar biologic, pharmacologic, or toxicologic activities. Additional information about the significance of toxicity at low dosea has been obtained from the few very low-dose animal carcinogenicity studies that have been conducted, namely, aflatoxin (5-fold dose spread) (6),1,3-butadiene (200-fold) (7,8), and nitrosamines (500-fold) (!+ll).It is noteworthythat, for these substances, carcinogenicactivity was found over a wide range of doses, not just the high dose, and at doses with little apparent toxicity. The generalizability of these findings is unknown because as yet few such studies have been reported. Very low dose toxicity studies have been conducted also with tetrachlorodibenzodioxin (TCDD)(12),but the relevance of receptor kinetic and other data to cancer formation is still uncertain. The hypotheses that the toxic effects of chemicals might contribute to increased rates of mutations or to decreased cellular repair rates are compelling. One caution, while research continues, is that all chemical substances are toxic at some doses but half of those tested thus far by NTP do not exhibit carcinogenic activity.
MTD Not? It has been stated in the lay press that two-thirds of the NTP carcinogens would not be positive if the MTD were not used. That conclusion is a misinterpretation of data
740 Chem. Res. Toxicol., Vol. 5, No. 6,1992
presented by Haseman (IO)which applied to tumors rather than to chemicals in a small sample of 13 rodent carcinogens. In a larger series of NTP studies ( 4 )on 52 chemical carcinogens, including the 13 evaluated by Haseman, 34 (65%) would still have been positive without the MTD and 15 of the 18 not showing significant tumor responses at doses below the MTD nevertheless had numerically elevated tumor rates at these doses relative to controls. If the analysis were restricted to substances that require multiple sex-species effects for classification of carcinogenicity, 31 of 38 carcinogens (82%) would have been detected without the MTD. One must remember that removing the MTD eliminates one-third of the data, which reduces the power of any statistical test regardless of dose considerations. In most earlier NTP and National Cancer Institute (NCI) studies animals were exposed only a t the MTD and MTDI2, but more recent studies have also exposed animals at MTD/4. Among 38 positive resonses in sex-and speciesspecific studies, 23 (61%) would have been positive if MTD/4 had been the largest dose. Seven of the 23 would have detected all site-specific responses, and the other 16 would have detected some, but not all, site-specificeffects, indicating how low experimental doses can be in standard bioassays and still provide useful doseresponse information (i.e., stillresult in responses that are distinguishable from background).
Are We There Yet? Animal carcinogenicity studies comprise the only presently available definitive approach to predict carcinogenic hazards and prevent unnecessary human exposures, but they are not entirely satisfactory. In particular, animal studies are resource intensive and do not provide enough information by themselves to provide the basis for more than conservative public health measures. Epidemiologic studies using people who have already been exposed to carcinogens is not a satisfactory alternative. To a great extent, however, the predictive capabilities of animal carcinogenicity studies are now improving through the addition of ancillary studies and improvements in the science base. As part of the preliminary toxicity investigations, studies of the disposition of the test substance (uptake, distribution, metabolism, storage, detoxification, excretion) are usually performed to aid in the selection of the test species, the route of exposure, the choice of delivery vehicle, and the dose regimen for the long-term studies. When unexpected results are obtained in the long-term studies (such as sex differences, unpredicted tissue sites, or unusual tumor types), follow-up targeted studies many be conducted to confiim the original observations, to seek explanations through mechanistic studies, and to examine doseresponse relationships. The design of a program of research studies for the substance in question is simplified when dealing with a member of a group of substancesthat have been so well studied already that physiologic, pharmacologic, and pathologic effects can be anticipated and measured. The major limiting factor for trans-species extrapolations, however, continues to be the relative paucity of comparable information from exposed humans. Where Next? Until relatively recently, carcinogenicity studies in animals and people have been largely observational.
Griesemer
Epidemiologic studies have provided information about the relative importance of "early" or "late" exposures, and animal studies have indicated stages of exposure and response labeled operationally as "initiation", "promotion", "progression", and the like. While helpful in telling experimentalists generally what to look for and when to look, the underlying scientific basis for the observations has been largely elusive. The situation is now rapidly changing. New concepts and techniques of molecular biology and genetics applied to cancer patients or to experimentalanimals are providing testable hypotheses about the relations between exposure to environmental substances and subsequent carcinogenesis. It is now possible to begin to identify and measure the critical and rate-affecting events in the multistage carcinogenic processes. The exciting result is the convergence of information derived from humans and experimental animals that is beginning to provide a firm basis for interspecies extrapolations. It can be anticipated that default trans-species and low-dose extrapolationswill be replaced progressively by biologicallybased, validated models.
Predictions without Animal Studies The NTP's systematic collection of animal carcinogenicity data with standardized methods and quality controls has made it possible to evaluate the utility of this approach for scientific and public health purposes. Among the constructive criticisms from our own staff is the suggestion that it might be possible now to make sufficiently accurate predictions about the potential carcinogenicitysubstances that animal studies may no longer be needed. Accordingly, we have evaluated the data available on 44 NTP long-term animal studies that were still in progress. Using our knowledge of chemical structural considerations, genotoxicity, and biologic activity, we made predictions about the outcome of the carcinogenicity studies. The publication of our predictions (13)included a challenge to others to make their predictions for the same substances. Five other groups did so, publishing their predictions in the same journal. When the animal studies are completed later this year, we plan a workshop to break the code and learn what we can, not only about our collective abilities to predict carcinogenicity,but about the basis for decisionmaking and the types of data useful for making predictions. Summing Up High-dose selection for animal carcinogenicity studies has a rational basis and provides optimal sensitivity to identify and characterize carcinogenic hazards. The results of animal carcinogenicity studies serve as a frame of reference for subsequent investigations to obtain the information needed to protect people from unnecessary exposure to hazardous substances. Alternative methods and approaches to high-dose studies and to the animal studies themselves are being vigorously pursued by NTP, but as yet there is no satisfactory substitute for long-term animal carcinogenicity studies as presently performed. Note Added in Proof. While this paper was being prepared, the NTP sponsored another in a series of public meetings to seek adviceabout improving ita programs (Fed. Regist. 57, 31721-31730, July 17, 1992). Advice is welcome anytime. References (1) McConnell, E. E. (1989) The maximum tolerated dose: the debate. J.Am. Coll. Toxicol. 8,1115-1120.
Forum (2) Montesano,R., Bartsch, H., Vainio, H., Wilbourn, J., and Yamasaki, H. (1986) Long-term and short-term assays for carcinogens: A critical appraisal, IARC Scientific Publications No. 83,IARC, Lyon. (3) Gart, J. J.,Krewski, P. N., Tarone, R. E., and Wahrendorf, J. (1986) Experimental Design. In StatisticaE methods in cancer research-Volume III-The design and analysis of long-term animal erperiments, IARC Scientific Publications No. 79, pp 21-56, Oxford University Prees, Oxford, U.K. (4) Hoel, D. G., Haseman, J. K., Hogan, M. D., Huff, J., and McConnell, E. E. (1988) The impact of toxicity on carcinogenicity studies: implications for risk aseessment. Carcinogenesis 9, 2045-2052. (5) Tennant, R. W., Elwell, M. R., Spalding, J. W., and Griesemer, R. A. (1991) Evidence that toxic injury is not always associated with induction of chemical carcinogenesis. Mol. Carcinog. 4, 42C-440. (6) Staffa, J. A., and Mehlman, M. A. (1980) Innovations in cancer risk asseesment (ED01study). J. Enuiron. Pathol. Toricol. 3 (3) (Special Issue). (7) Melnick, R. L., Huff, J., Chou, B. J., and Miller, R. A. (1990) Carcinogenicity of 1,a-butadienein C57BL/6 X C3H F1 mice at low exposure concentrations. Cancer Res. 50,65926699. (8) Toxicology and carcinogenesis studies of 1,3-butadiene in B6C3F1 mice (1991) NTP Technical Report No. 434, NIH Publication No.
Chem. Res. Toxicol., Vol. 5, No.6, 1992 741 92-3165 (in press). (9) Peto, R., Gray, R., Brantom, P., and Grasso, P. (1991)Effwta on 4080 rats of chronic ingestion of N-nitrosodiethylamine: a detailed dose-response study. Cancer Res. 51, 6415-6451. (10) Peto, R., Gray, R., Brantom, P., and Grasso, P. (1991)Dose and time relationships for tumor induction in the liver and esophagus of 4080 inbred rata by chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine. Cancer Res. 51,6462-6469. (11) Gray, R., Peto, R., Brantom, P., and Grasso, P. (1991) Chronic nitrosamine ingestion in 1040 rodents: the effect of the choice of nitrosamine, the species studied and the age of starting exposure. Cancer Res. 51,6470-6491. (12) Tritscher, A. M., Goldstein, J. A,, Portier, C. J., McCoy, Z., Clark, G. C., and Lucier, G. W. (1992) Doseresponse relationships for chronicexposureto 2,3,7,S-tetrachlorodibenmpdioxin in arat tumor promotion model: quantitication and immunolocalizationof CYPlAl and CYPlA2 in the liver. Cancer Res. 52,3436-3442. (13) Tennant, R. W., Spalding, J., Stasiewicz, S., and Ashby, J. (1990) Prediction of the outcome of rodent carcinogenicity bioassays currently being conducted on 44 chemicals by the National Toxicology Program. Mutagenesis 5, 3-14.
