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The role of genetic toxicology in drug safety evaluation
REGULATORY
TOXICOLOGY
AND
PHARMACOLOGY
2, 177- 183 (1982)
The Role of Genetic Toxicology
in Drug Safety Evaluation
Prepared by the Drug Safety Subsection Steering Committee Pharmaceutical Manufacturers Association
M. SCHACH VON WITTENAU, PH.D.,
of the
VICE CHAIRMAN
Vice President, Safety Evaluation. Pfizer Central Research
AND JOSEPH E. LEBEAU, DVM,
CHAIRMAN
Director, Pre-Clinical Research, Merrell Dow Pharmaceuticals
Received March 198.2
Investigation of the mutagenic properties of chemicals is an exciting field of research which has led to many important discoveries and a better understanding of how chemicals interact with genetic material. The discipline of genetic toxicology, however, is very much in the developmental stage. Test methodologies are still being validated, both for their reproducibility and for the relevance to safety assessment. In vitro experiments can disclose a potential for toxicological effects (such as oncogenic, teratogenic, and reproductive effects) which may be linked to mutagenic mechanisms, but they cannot prove that a compound will pose those risks to mammals. Once effects in whole animals have been observed, however, genetic toxicology can help define mechanisms and make valuable contributions to risk assessment. If results from animal studies have shown that no risk exists, investigations of mutagenic activity are not likely to provide any relevant information. Used as a screen, mutagenicity testing has value but is of uncertain predictability as to toxicity in whole animals.
The realization that chemicals can affect genetic material has been followed by intensive studies in many laboratories aspiring to understand mechanisms and implications. The impressive gain in knowledge provides the basis for the relatively new discipline of genetic toxicology. While early on the primary concern had been focused on possible impacts on the human gene pool, i.e., alterations in germ cells resulting in changes in hereditary characteristics, the apparent relationship between mutagenic activity and initiation of neoplasms caused investigators to explore the mutation theory for the etiology of oncogenicity. Today, the term mutagenicity is understood not only as applying to effects in germ cells, but also as encompassing 117 0273-2300/82/030177-07$02.00/O Copyright Q 1982 by Academic Pm.% Inc. AII rights of reproduction in my form resmd.
178
PHARMACEUTICAL
MANUFACTURERS
ASSOCIATION
alterations in the genetic material of somatic cells. Discussions of possible health consequences of exposure to mutagenic agents generally address the possibility that such agents might cause cancer, induce fetal loss, result in birth defects, and be capable of affecting those neurological and metabolic diseases known to be genetic in origin (1). Recognizing that genetic toxicology can make a contribution to the safety assessment of chemicals, regulatory agencies concerned with human health expect submission of data that define the mutagenic activity of chemicals (2). As such expectations inevitably are translated into testing requirements, it appears timely and appropriate for the PMA to define the role genetic toxicology can play in the safety assessment of chemicals intended to be introduced as drugs. Techniques used by genetic toxicologists span the range from observations in man to experiments with subcellular components of living organisms. Many assay procedures are appealing because they incur a relatively low cost in time and resources, and are designed to provide clear information regarding a compound’s activity in the particular system selected for study. Often insight into the event causing toxicity is provided at the molecular level, leading to an understanding of the relationship between the structure of a chemical and the observed toxicity. As all living organisms depend upon the integrity of their genetic material, interaction of a chemical with DNA observed in one test system frequently is perceived as having meaning to all forms of life, including man. This generalization does, however, encounter criticism; the models employed are phylogenetically, biochemically, and methodologically so diverse that the relevance of some to the assessment of human health risk is far from obvious. A positive response in a particular test system establishes genetic activity under the conditions of that experiment, but such observation, unless made in man, does not indicate more than a potential to damage genetic material in man. This complication is inherent in the use of any surrogate, and biologists attempting to discover effective drugs have learned to deal with the uncertainties associated with extrapolations to man. A balanced interpretation of genetic toxicology data, however, often is frustrated by forces from outside science; insofar as testing for mutagenic activity is seen as an endeavor to prevent cancer, or to protect future generations of mankind from genetic liability, a well-reasoned interpretation of test data can become encumbered by the emotional need to perceive absolute safety. While the abundance of available methodologies permits in many instances a credible estimate of a chemical’s potential to harm the genetic material in man, the danger is real that positive and negative data will be weighed on the basis not of sound science, but out of fears. Mutagenic chemicals were not first introduced into man’s environment by technological activities, but have always existed, for example, in our food supplies (3), and are even generated by mammals themselves (4). Heritable diseases are recognized in man, but so far there is no evidence that chemicals are the cause of such diseases, despite the specter having been raised frequently. On the other hand, nothing is known that unequivocally rules out such a possibility. In view of these complexities, testing for mutagenic activity can be a support or a hindrance to safety evaluation, depending upon how responsibly this new technology is used. An assessment of the utility of genetic toxicology data for the safety evaluation of chemicals must consider the degree to which such information has been shown to predict effects in man or at least in mammals, the reliability of the techniques used in testing for mutagenicity, and other toxicological information available at
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IN DRUG SAFETY EVALUATION
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the time such data are obtained. Interspecies and intercellular differences in susceptibilities to mutagenic agents encumber the utility of models. The finding in a system other than the whole animal that a chemical exhibits mutagenic activity does not allow the conclusion that such activity will take place in vivo and that this potential to do harm will be expressed and cause any of the adverse effects cited. Such information can only be obtained from animal studies conducted under realistic conditions. Deliberate testing is not possible in man, and animals must be used as surrogates. For the purpose of assessing health risk, models are ordered in a hierarchy of relevance. One may wish to envision a scheme of test systems as a pyramid, with man at the apex, in vitro studies of molecular interactions at the base, and animal experiments in between, each occupying tiers according to their established relevance as predictors of human health hazards. Each test system employed must be characterized regarding its accuracy in predicting effects in man and its reliability to give reproducible results. While the latter is relatively easily addressed, the validity of a model as a surrogate for man often is difficult to establish. Nevertheless, unless this is accomplished, the usefulness of the test system to safety evaluation is limited. An adverse effect observed in a given model merely suggests the potential for detrimental activity at a higher level. At that higher tier, a mutagenic response strengthens the suspicion of risk, but if that system is known to adequately detect hazards to man, then a negative response removes such suspicion; the mutagenic activity apparently is nullified by the compensatory or protective mechanisms in the higher tier model. Test systems should not be selected on the basis of “which test gives the most positive results,” but on relevance. The former approach leads to findings in search of a meaning, the latter uses a manifestation of toxicity observed in animal or man as a reference point, and searches for a test that will predict such toxicity with the greatest accuracy possible. Unless safety evaluation is subjected to that discipline of thought, it will be unproductive by raising but not putting to rest suspicions of disaster, and by failing to distinguish real from imagined risks. Mutagenic chemicals often are animal carcinogens. Several surveys have concluded that a mutagen has a higher probability of being carcinogenic than does a nonmutagen (5). The mechanistic relationship is not well understood, and it is clear that genetic activity does not necessarily presage oncogenicity. Conversely, not all apparent carcinogens can be shown to possess mutagenic activity, a fact whose recognition has led to the distinction between genotoxic and nongenotoxic carcinogens and the proposal to differentiate between these for the purpose of risk assessment (6). In view of this imperfect correlation between mutagenicity and oncogenicity, the assessment of carcinogenic risk to man will continue to have to rely on oncogenicity studies in those mammals which are accepted as surrogates for man. Once oncogenic activity has been detected, mutagenicity studies can reveal whether this is related to genotoxic or nongenotoxic mechanisms. Such information is very valuable to have because considerations of the mode of action will facilitate the extrapolation of data from animal to man and from the laboratory to practical use. Mutagenicity testing plays a similar role in assessing the risk of impaired reproductive performance, or of functional alterations of offspring. A finding of mutagenic activity in an artificial test system does not predict with certainty toxicity in the whole animal. Such toxicity may be caused by a variety of mechanisms, of which
180
PHARMACEUTICAL
MANUFACTURERS
ASSOCIATION
genetic activity is only one. Animal experiments will identify a potential hazard to man, and genetic toxicology data may provide insight into the molecular events that resulted in an adverse effect. The above discussion arrives at the position that a finding of mutagenicity in systems other than the whole mammal suggests only the possibility of harm to animals; that experiments with mammals must be relied upon when determining whether a potential risk exists for man; and that once such potential risk has been identified through animal experimentation, mutagenicity data are helpful to the interpretation of meaning. Induced mutations are perceived as deleterious because they are thought to have the potential for causing a multitude of adverse effects. Genetic toxicology focuses on specific events such as point mutations, chromosomal aberrations, etc. “Classical” toxicology, which is designed to detect many of their detrimental consequences, such as cancer, fetal wastage, and fetal malformations, will observe such toxicity even if caused by mechanisms other than mutagenicity. It is difficult to accept the assumption that the expression of mutagenic activity will be confined entirely to that genetic material whose alteration gives rise only to heritable mutations not observable by “classical” experiments, and no example supporting that assumption is known. While such alterations theoretically may occur, it is highly unlikely that they happen exclusively. Tests for mutagenic activity offer the opportunity to the sponsor of a potential drug to gauge the probability of a chemical showing serious toxicity in more costly experiments. The development of a drug consumes vast quantities of money, time, and scientific resources. Often a choice has to be made as to which chemical to select for development. Under those circumstances, it is useful to know whether or not a chemical can damage genetic material and has the pomtiui to cause in mammals toxicity linked to mutagenic mechanisms. Many decision making processes are facilitated by data which indicate whether or not such potential exists, even if it is not known that such potential will be expressed in whole animals. The use of mutagenicity testing as a screen can be justified on the basis of economics. While the danger is real that a potentially valuable product is discarded merely on suspicion of relevant toxicity, evaluation in an appropriate battery of mutagenicity tests should minimize that risk. If a chemical is perceived as a potentially valuable drug, followup studies can provide a clearer perspective. Limited investigations of the toxicology of a chemical may show that detrimental effects resulting from nongenetic mechanisms are of little concern either because the relevant pharmacologic potency of the chemical is very low and/or because human exposure to the compound is quite limited. Tissue residues of veterinary drugs are cited as an example to which this approach may be applied. It then appears reasonable to demonstrate nonexistence of mutagenic activity and thus absence of the potential to do harm via a genotoxic mechanism. Up to this point this discussion has focused on the concepts which determine the proper utilization of genetic toxicology in safety evaluation, without addressing the state of the art of mutagenicity testing. The enthusiasm for this relatively new technology has led to a cornucopia of more than 100 tests (7), of which some have not been replicated in an adequate number of laboratories. As several panels, such as those established by the International Commission for Protection Against Environmental Mutagens and Carcinogens (ICPEMC), by the National Toxicology Program,
GENETIC TOXICOLOGY
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and by the Environmental Protection Agency’s Gene Tox Program, are still attempting to define the utility and practicality of some of these procedures, PMA will not take a position on individual techniques at this time. PMA proposes instead several general criteria which should be observed, if experiments investigating genetic activity are to be used not just in the context of exploratory research, but also for the saCety assessment of drugs. The impact toxicology data have on the availability of a drug to the physician dictates that experimental procedures used in safety assessment are well controlled, soundly interpreted, and that results can be replicated by different laboratories. As long as this state of maturity has not been achieved, research into mutagenic effects must remain exploratory. As the traditional toxicology studies detect harmful consequences of exposure to chemicals capable of genetic activity, the patient meanwhile is protected. The validity of the various tests as predictors of human hazard must be more firmly established than has been done to date. The burden of proof of validity rests with the proponent of a model. A procedure, which is capable only of raising suspicion, is useless unless a test of a higher tier can provide the definitive answer, in which case it may often be advisable to use the latter initially. As stated before, it appears reasonable to assume that the closer the test system is to the whole animal, the more meaningful is the information obtained. There is general agreement that no single assay will suffice to detect mutagenic activity of chemicals, and that a battery of tests is needed (8). It appears that as yet the ideal composition of such a battery has not been defined for safety assessment purposes. The spectrum of available methods measures various endpoints such as molecular alterations of DNA and cytological observation of gross chromosomal damage. It includes tests such as sister chromatid exchange, which reveal genetic activity that has not yet been shown to result in a detrimental health effect, and procedures such as the dominant lethal assay, in which a positive finding, if a consequence of genetic activity, is assumed to have high relevance to human risk. The percentage of chemicals identified as possessing genetic activity varies greatly with the tests applied. Generally, the closer the test system is to the whole animal, the less “sensitive” the assay appears to be, yet the more relevant it seems to be to safety assessment. On the other hand, the models thought to be highly relevant must be able to detect chemicals that are mutagenic in man. A recent survey of 614 chemicals tested for carcinogenicity and evaluated by a panel of experts revealed that relatively few have been subjected to a battery of mutagenicity assays as judged by the open literature (9). This indicates that much more work is needed before the relevance to human health of most mutagenicity tests can be assessed. Subjecting only a few carefully selected compounds to a given test procedure can be misleading regarding the utility of an assay. A positive finding in an in vitro assay for genetic activity raises the suspicion that such activity may be manifested in animals as somatic and/or germ cell mutations. To gain perspective on such observations in terms of human risk, further expetiments are needed to assesswhether or not this potential for harm actually is expressed in mammals. It is generally believed that in whole animals genotoxicity in somatic cells leads to death of some cells and/or to cancer. Absence of a somatic effect ahO indicates that germ cells which appear to be better protected than somatic cells may not be affected. In a recent extensive literature review (20) a total of 76 compounds
182
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MANUFACTURERS
ASSOCIATION
were found to have been tested for chromosomal effects in viva in both somatic and germ cells. Most notable is the fact that no compound was found to produce positive results in germ cell assays and negative results in somatic cells, i.e., every compound positive in germ cells was also positive in somatic cells. Furthermore, a large proportion of active compounds (40%) were negative in germ cells but positive in somatic cells. Assessment of the consequences in man of radiation also suggests a higher susceptibility of somatic cells than of germ cells (21). CONCLUSIONS Investigations of the mutagenic properties of chemicals is an exciting field of research which has led to many important discoveries and a better understanding of how chemicals interact with genetic material. The discipline of genetic toxicology is very much in a developmentaI stage. Test methodologies are still being validated, both for their reproducibility and for their relevance to safety assessment. Several scientific panels currently are involved in that validation process. In addition, technology for assessment of genotoxicity will continue to improve in the future; it is therefore premature for us or for regulating bodies to take a formal position regarding individual procedures. Conceptually, tests for mutagenicity in drug research are seen as aiding in the understanding of observations made in toxicology experiments usually conducted before a drug is approved. Studies investigating possible oncogenicity, teratogenicity, and impaired reproductive performance are designed to detect among others those consequences mutagenic activity is suspected of having in whole animals. As demonstrated absence of such consequences makes moot the question of potentiul for doing harm, there usually will be no regulatory need for mutagenicity data when negative oncogenic, teratogenic, and reproductive data are available. Under certain circumstances, however, it appears scientifically sound to substitute a comprehensive battery of mutagenicity data for oncogenicity experiments. Such will be the case when low human exposure and knowledge of the pharmacologic activity of the chemical indicates that an oncogenic hazard could only be envisioned if the compound were to possess genotoxic properties. It may be advantageous to evaluate chemicals for genetic activity before committing large resources to their development. A positive finding may suggest the possibility of irreversible toxicity in man, with a probability which will depend upon the test used, but which has not yet been defined for any method. Before employing a battery of procedures as a screen, the complementary as well as the redundant nature of the selection should be defined. Whole animal models are likely to be more relevant than in vitro procedures. While the latter generally will be less expensive, they also are expected to identify more chemicals as mutagens, though not as mutagens in mammals, than the former. The selection of an appropriate battery of tests will consequently depend upon the objectives. A trade-off will exist between relevancy to human health and ease of experimentation, although a high price in unnecessarily discarded chemicals may be paid when apparently inexpensive tests are used to indict compounds without subsequently subjecting them to procedures appropriate for human risk assessment.
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REF%RENCES I. PREEsE,E. (197 1). In ChemicalMutagens: Principles andMethodsfor TheirDetection (A. Hollaender, Ed.), Vol. 1, p. 14. Plenum, New York. 2. Examples include Department of Health and Social Security (U.K.), Environmental Protection Agency (U.S.), and Health Protection Branch (Canada), although the latter does not routinely demand that such data are generated. 3. MILLER, E. C., et aI. (1979). NaturaIIy Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis. Univ. Park Press, Baltimore. 4. TANNENBAUM, S. R., ibid., p. 211. 5. BRUSICK, D. (1980). In Principles of Genetic Toxicology, p. 5% Plenum, New York. 6. WEISBURGER,J. H., AND WILLIAMS, G. M. (1977). In Structural Correlates of Carcinogenesis and Mutagenesis (I. M. Asher and C. Zervos, Eds.), p. 45. Office of Science, IDA; WILLIAMS, G. M. (1979). Review of in vitro test systems using DNA damage and repair for screening of chemical carcinogens. .L Assoc. Ofl Anal. Chem. 62, 857; Health Council of The Netherlands. (1980). Report on the Evaluation of the Carcinogenicity of Chemical Substances. 7. HOLLSTEIN, M. C. (1979). In Banbuty Report: 2. Mammalian CeB Mutagenesis (V. K. McEhkeny and S. Abrahamson, Eds.), p. 431. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 8. BRUSICK, D. (1980). In Principles of Genetic Toxicology, Chap. 4. Plenum, New York. 9. SODERMAN, J. V. Handbook of Zdenttfied Carcinugens and Noncarcinogens. CRC Press, Boca Raton, in press. IO. HOLDEN, H. E., Comparison of somatic and germ cell models for cytogenetic screening. .Z. Appt. Toxicol., in press; see also Science (1982), 215,458. II. SCHULL, W., OTAKE, M. AND NEEL, J. (1981). Genetic effects of the atomic bombs: A reappraisal. Science 213, 1220.
TOXICOLOGY
AND
PHARMACOLOGY
2, 177- 183 (1982)
The Role of Genetic Toxicology
in Drug Safety Evaluation
Prepared by the Drug Safety Subsection Steering Committee Pharmaceutical Manufacturers Association
M. SCHACH VON WITTENAU, PH.D.,
of the
VICE CHAIRMAN
Vice President, Safety Evaluation. Pfizer Central Research
AND JOSEPH E. LEBEAU, DVM,
CHAIRMAN
Director, Pre-Clinical Research, Merrell Dow Pharmaceuticals
Received March 198.2
Investigation of the mutagenic properties of chemicals is an exciting field of research which has led to many important discoveries and a better understanding of how chemicals interact with genetic material. The discipline of genetic toxicology, however, is very much in the developmental stage. Test methodologies are still being validated, both for their reproducibility and for the relevance to safety assessment. In vitro experiments can disclose a potential for toxicological effects (such as oncogenic, teratogenic, and reproductive effects) which may be linked to mutagenic mechanisms, but they cannot prove that a compound will pose those risks to mammals. Once effects in whole animals have been observed, however, genetic toxicology can help define mechanisms and make valuable contributions to risk assessment. If results from animal studies have shown that no risk exists, investigations of mutagenic activity are not likely to provide any relevant information. Used as a screen, mutagenicity testing has value but is of uncertain predictability as to toxicity in whole animals.
The realization that chemicals can affect genetic material has been followed by intensive studies in many laboratories aspiring to understand mechanisms and implications. The impressive gain in knowledge provides the basis for the relatively new discipline of genetic toxicology. While early on the primary concern had been focused on possible impacts on the human gene pool, i.e., alterations in germ cells resulting in changes in hereditary characteristics, the apparent relationship between mutagenic activity and initiation of neoplasms caused investigators to explore the mutation theory for the etiology of oncogenicity. Today, the term mutagenicity is understood not only as applying to effects in germ cells, but also as encompassing 117 0273-2300/82/030177-07$02.00/O Copyright Q 1982 by Academic Pm.% Inc. AII rights of reproduction in my form resmd.
178
PHARMACEUTICAL
MANUFACTURERS
ASSOCIATION
alterations in the genetic material of somatic cells. Discussions of possible health consequences of exposure to mutagenic agents generally address the possibility that such agents might cause cancer, induce fetal loss, result in birth defects, and be capable of affecting those neurological and metabolic diseases known to be genetic in origin (1). Recognizing that genetic toxicology can make a contribution to the safety assessment of chemicals, regulatory agencies concerned with human health expect submission of data that define the mutagenic activity of chemicals (2). As such expectations inevitably are translated into testing requirements, it appears timely and appropriate for the PMA to define the role genetic toxicology can play in the safety assessment of chemicals intended to be introduced as drugs. Techniques used by genetic toxicologists span the range from observations in man to experiments with subcellular components of living organisms. Many assay procedures are appealing because they incur a relatively low cost in time and resources, and are designed to provide clear information regarding a compound’s activity in the particular system selected for study. Often insight into the event causing toxicity is provided at the molecular level, leading to an understanding of the relationship between the structure of a chemical and the observed toxicity. As all living organisms depend upon the integrity of their genetic material, interaction of a chemical with DNA observed in one test system frequently is perceived as having meaning to all forms of life, including man. This generalization does, however, encounter criticism; the models employed are phylogenetically, biochemically, and methodologically so diverse that the relevance of some to the assessment of human health risk is far from obvious. A positive response in a particular test system establishes genetic activity under the conditions of that experiment, but such observation, unless made in man, does not indicate more than a potential to damage genetic material in man. This complication is inherent in the use of any surrogate, and biologists attempting to discover effective drugs have learned to deal with the uncertainties associated with extrapolations to man. A balanced interpretation of genetic toxicology data, however, often is frustrated by forces from outside science; insofar as testing for mutagenic activity is seen as an endeavor to prevent cancer, or to protect future generations of mankind from genetic liability, a well-reasoned interpretation of test data can become encumbered by the emotional need to perceive absolute safety. While the abundance of available methodologies permits in many instances a credible estimate of a chemical’s potential to harm the genetic material in man, the danger is real that positive and negative data will be weighed on the basis not of sound science, but out of fears. Mutagenic chemicals were not first introduced into man’s environment by technological activities, but have always existed, for example, in our food supplies (3), and are even generated by mammals themselves (4). Heritable diseases are recognized in man, but so far there is no evidence that chemicals are the cause of such diseases, despite the specter having been raised frequently. On the other hand, nothing is known that unequivocally rules out such a possibility. In view of these complexities, testing for mutagenic activity can be a support or a hindrance to safety evaluation, depending upon how responsibly this new technology is used. An assessment of the utility of genetic toxicology data for the safety evaluation of chemicals must consider the degree to which such information has been shown to predict effects in man or at least in mammals, the reliability of the techniques used in testing for mutagenicity, and other toxicological information available at
GENETIC TOXICOLOGY
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the time such data are obtained. Interspecies and intercellular differences in susceptibilities to mutagenic agents encumber the utility of models. The finding in a system other than the whole animal that a chemical exhibits mutagenic activity does not allow the conclusion that such activity will take place in vivo and that this potential to do harm will be expressed and cause any of the adverse effects cited. Such information can only be obtained from animal studies conducted under realistic conditions. Deliberate testing is not possible in man, and animals must be used as surrogates. For the purpose of assessing health risk, models are ordered in a hierarchy of relevance. One may wish to envision a scheme of test systems as a pyramid, with man at the apex, in vitro studies of molecular interactions at the base, and animal experiments in between, each occupying tiers according to their established relevance as predictors of human health hazards. Each test system employed must be characterized regarding its accuracy in predicting effects in man and its reliability to give reproducible results. While the latter is relatively easily addressed, the validity of a model as a surrogate for man often is difficult to establish. Nevertheless, unless this is accomplished, the usefulness of the test system to safety evaluation is limited. An adverse effect observed in a given model merely suggests the potential for detrimental activity at a higher level. At that higher tier, a mutagenic response strengthens the suspicion of risk, but if that system is known to adequately detect hazards to man, then a negative response removes such suspicion; the mutagenic activity apparently is nullified by the compensatory or protective mechanisms in the higher tier model. Test systems should not be selected on the basis of “which test gives the most positive results,” but on relevance. The former approach leads to findings in search of a meaning, the latter uses a manifestation of toxicity observed in animal or man as a reference point, and searches for a test that will predict such toxicity with the greatest accuracy possible. Unless safety evaluation is subjected to that discipline of thought, it will be unproductive by raising but not putting to rest suspicions of disaster, and by failing to distinguish real from imagined risks. Mutagenic chemicals often are animal carcinogens. Several surveys have concluded that a mutagen has a higher probability of being carcinogenic than does a nonmutagen (5). The mechanistic relationship is not well understood, and it is clear that genetic activity does not necessarily presage oncogenicity. Conversely, not all apparent carcinogens can be shown to possess mutagenic activity, a fact whose recognition has led to the distinction between genotoxic and nongenotoxic carcinogens and the proposal to differentiate between these for the purpose of risk assessment (6). In view of this imperfect correlation between mutagenicity and oncogenicity, the assessment of carcinogenic risk to man will continue to have to rely on oncogenicity studies in those mammals which are accepted as surrogates for man. Once oncogenic activity has been detected, mutagenicity studies can reveal whether this is related to genotoxic or nongenotoxic mechanisms. Such information is very valuable to have because considerations of the mode of action will facilitate the extrapolation of data from animal to man and from the laboratory to practical use. Mutagenicity testing plays a similar role in assessing the risk of impaired reproductive performance, or of functional alterations of offspring. A finding of mutagenic activity in an artificial test system does not predict with certainty toxicity in the whole animal. Such toxicity may be caused by a variety of mechanisms, of which
180
PHARMACEUTICAL
MANUFACTURERS
ASSOCIATION
genetic activity is only one. Animal experiments will identify a potential hazard to man, and genetic toxicology data may provide insight into the molecular events that resulted in an adverse effect. The above discussion arrives at the position that a finding of mutagenicity in systems other than the whole mammal suggests only the possibility of harm to animals; that experiments with mammals must be relied upon when determining whether a potential risk exists for man; and that once such potential risk has been identified through animal experimentation, mutagenicity data are helpful to the interpretation of meaning. Induced mutations are perceived as deleterious because they are thought to have the potential for causing a multitude of adverse effects. Genetic toxicology focuses on specific events such as point mutations, chromosomal aberrations, etc. “Classical” toxicology, which is designed to detect many of their detrimental consequences, such as cancer, fetal wastage, and fetal malformations, will observe such toxicity even if caused by mechanisms other than mutagenicity. It is difficult to accept the assumption that the expression of mutagenic activity will be confined entirely to that genetic material whose alteration gives rise only to heritable mutations not observable by “classical” experiments, and no example supporting that assumption is known. While such alterations theoretically may occur, it is highly unlikely that they happen exclusively. Tests for mutagenic activity offer the opportunity to the sponsor of a potential drug to gauge the probability of a chemical showing serious toxicity in more costly experiments. The development of a drug consumes vast quantities of money, time, and scientific resources. Often a choice has to be made as to which chemical to select for development. Under those circumstances, it is useful to know whether or not a chemical can damage genetic material and has the pomtiui to cause in mammals toxicity linked to mutagenic mechanisms. Many decision making processes are facilitated by data which indicate whether or not such potential exists, even if it is not known that such potential will be expressed in whole animals. The use of mutagenicity testing as a screen can be justified on the basis of economics. While the danger is real that a potentially valuable product is discarded merely on suspicion of relevant toxicity, evaluation in an appropriate battery of mutagenicity tests should minimize that risk. If a chemical is perceived as a potentially valuable drug, followup studies can provide a clearer perspective. Limited investigations of the toxicology of a chemical may show that detrimental effects resulting from nongenetic mechanisms are of little concern either because the relevant pharmacologic potency of the chemical is very low and/or because human exposure to the compound is quite limited. Tissue residues of veterinary drugs are cited as an example to which this approach may be applied. It then appears reasonable to demonstrate nonexistence of mutagenic activity and thus absence of the potential to do harm via a genotoxic mechanism. Up to this point this discussion has focused on the concepts which determine the proper utilization of genetic toxicology in safety evaluation, without addressing the state of the art of mutagenicity testing. The enthusiasm for this relatively new technology has led to a cornucopia of more than 100 tests (7), of which some have not been replicated in an adequate number of laboratories. As several panels, such as those established by the International Commission for Protection Against Environmental Mutagens and Carcinogens (ICPEMC), by the National Toxicology Program,
GENETIC TOXICOLOGY
IN DRUG SAFETY EVALUATION
181
and by the Environmental Protection Agency’s Gene Tox Program, are still attempting to define the utility and practicality of some of these procedures, PMA will not take a position on individual techniques at this time. PMA proposes instead several general criteria which should be observed, if experiments investigating genetic activity are to be used not just in the context of exploratory research, but also for the saCety assessment of drugs. The impact toxicology data have on the availability of a drug to the physician dictates that experimental procedures used in safety assessment are well controlled, soundly interpreted, and that results can be replicated by different laboratories. As long as this state of maturity has not been achieved, research into mutagenic effects must remain exploratory. As the traditional toxicology studies detect harmful consequences of exposure to chemicals capable of genetic activity, the patient meanwhile is protected. The validity of the various tests as predictors of human hazard must be more firmly established than has been done to date. The burden of proof of validity rests with the proponent of a model. A procedure, which is capable only of raising suspicion, is useless unless a test of a higher tier can provide the definitive answer, in which case it may often be advisable to use the latter initially. As stated before, it appears reasonable to assume that the closer the test system is to the whole animal, the more meaningful is the information obtained. There is general agreement that no single assay will suffice to detect mutagenic activity of chemicals, and that a battery of tests is needed (8). It appears that as yet the ideal composition of such a battery has not been defined for safety assessment purposes. The spectrum of available methods measures various endpoints such as molecular alterations of DNA and cytological observation of gross chromosomal damage. It includes tests such as sister chromatid exchange, which reveal genetic activity that has not yet been shown to result in a detrimental health effect, and procedures such as the dominant lethal assay, in which a positive finding, if a consequence of genetic activity, is assumed to have high relevance to human risk. The percentage of chemicals identified as possessing genetic activity varies greatly with the tests applied. Generally, the closer the test system is to the whole animal, the less “sensitive” the assay appears to be, yet the more relevant it seems to be to safety assessment. On the other hand, the models thought to be highly relevant must be able to detect chemicals that are mutagenic in man. A recent survey of 614 chemicals tested for carcinogenicity and evaluated by a panel of experts revealed that relatively few have been subjected to a battery of mutagenicity assays as judged by the open literature (9). This indicates that much more work is needed before the relevance to human health of most mutagenicity tests can be assessed. Subjecting only a few carefully selected compounds to a given test procedure can be misleading regarding the utility of an assay. A positive finding in an in vitro assay for genetic activity raises the suspicion that such activity may be manifested in animals as somatic and/or germ cell mutations. To gain perspective on such observations in terms of human risk, further expetiments are needed to assesswhether or not this potential for harm actually is expressed in mammals. It is generally believed that in whole animals genotoxicity in somatic cells leads to death of some cells and/or to cancer. Absence of a somatic effect ahO indicates that germ cells which appear to be better protected than somatic cells may not be affected. In a recent extensive literature review (20) a total of 76 compounds
182
PHARMACEUTICAL
MANUFACTURERS
ASSOCIATION
were found to have been tested for chromosomal effects in viva in both somatic and germ cells. Most notable is the fact that no compound was found to produce positive results in germ cell assays and negative results in somatic cells, i.e., every compound positive in germ cells was also positive in somatic cells. Furthermore, a large proportion of active compounds (40%) were negative in germ cells but positive in somatic cells. Assessment of the consequences in man of radiation also suggests a higher susceptibility of somatic cells than of germ cells (21). CONCLUSIONS Investigations of the mutagenic properties of chemicals is an exciting field of research which has led to many important discoveries and a better understanding of how chemicals interact with genetic material. The discipline of genetic toxicology is very much in a developmentaI stage. Test methodologies are still being validated, both for their reproducibility and for their relevance to safety assessment. Several scientific panels currently are involved in that validation process. In addition, technology for assessment of genotoxicity will continue to improve in the future; it is therefore premature for us or for regulating bodies to take a formal position regarding individual procedures. Conceptually, tests for mutagenicity in drug research are seen as aiding in the understanding of observations made in toxicology experiments usually conducted before a drug is approved. Studies investigating possible oncogenicity, teratogenicity, and impaired reproductive performance are designed to detect among others those consequences mutagenic activity is suspected of having in whole animals. As demonstrated absence of such consequences makes moot the question of potentiul for doing harm, there usually will be no regulatory need for mutagenicity data when negative oncogenic, teratogenic, and reproductive data are available. Under certain circumstances, however, it appears scientifically sound to substitute a comprehensive battery of mutagenicity data for oncogenicity experiments. Such will be the case when low human exposure and knowledge of the pharmacologic activity of the chemical indicates that an oncogenic hazard could only be envisioned if the compound were to possess genotoxic properties. It may be advantageous to evaluate chemicals for genetic activity before committing large resources to their development. A positive finding may suggest the possibility of irreversible toxicity in man, with a probability which will depend upon the test used, but which has not yet been defined for any method. Before employing a battery of procedures as a screen, the complementary as well as the redundant nature of the selection should be defined. Whole animal models are likely to be more relevant than in vitro procedures. While the latter generally will be less expensive, they also are expected to identify more chemicals as mutagens, though not as mutagens in mammals, than the former. The selection of an appropriate battery of tests will consequently depend upon the objectives. A trade-off will exist between relevancy to human health and ease of experimentation, although a high price in unnecessarily discarded chemicals may be paid when apparently inexpensive tests are used to indict compounds without subsequently subjecting them to procedures appropriate for human risk assessment.
GENETIC TOXICOLOGY
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REF%RENCES I. PREEsE,E. (197 1). In ChemicalMutagens: Principles andMethodsfor TheirDetection (A. Hollaender, Ed.), Vol. 1, p. 14. Plenum, New York. 2. Examples include Department of Health and Social Security (U.K.), Environmental Protection Agency (U.S.), and Health Protection Branch (Canada), although the latter does not routinely demand that such data are generated. 3. MILLER, E. C., et aI. (1979). NaturaIIy Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis. Univ. Park Press, Baltimore. 4. TANNENBAUM, S. R., ibid., p. 211. 5. BRUSICK, D. (1980). In Principles of Genetic Toxicology, p. 5% Plenum, New York. 6. WEISBURGER,J. H., AND WILLIAMS, G. M. (1977). In Structural Correlates of Carcinogenesis and Mutagenesis (I. M. Asher and C. Zervos, Eds.), p. 45. Office of Science, IDA; WILLIAMS, G. M. (1979). Review of in vitro test systems using DNA damage and repair for screening of chemical carcinogens. .L Assoc. Ofl Anal. Chem. 62, 857; Health Council of The Netherlands. (1980). Report on the Evaluation of the Carcinogenicity of Chemical Substances. 7. HOLLSTEIN, M. C. (1979). In Banbuty Report: 2. Mammalian CeB Mutagenesis (V. K. McEhkeny and S. Abrahamson, Eds.), p. 431. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 8. BRUSICK, D. (1980). In Principles of Genetic Toxicology, Chap. 4. Plenum, New York. 9. SODERMAN, J. V. Handbook of Zdenttfied Carcinugens and Noncarcinogens. CRC Press, Boca Raton, in press. IO. HOLDEN, H. E., Comparison of somatic and germ cell models for cytogenetic screening. .Z. Appt. Toxicol., in press; see also Science (1982), 215,458. II. SCHULL, W., OTAKE, M. AND NEEL, J. (1981). Genetic effects of the atomic bombs: A reappraisal. Science 213, 1220.