Mutation Research, 33 (1975) 11--15 © Elsevier Publishing Company, Amsterdam -- Printed in The Netherlands
THE UTILITY OF SHORT-TERM TESTS FOR MUTAGENICITY IN THE TOXICOLOGICAL EVALUATION OF ENVIRONMENTAL AGENTS
FREDERICK J. D E SERRES Environmental Mutagenesis Branch, National Institute o f Environmental Health Sciences, P.O. B o x 12233, Research Triangle Park, N.C. 27709 (U.S.A.) (Received July 23rd, 1975) (Accepted AuguSt 25th, 1975)
About two years ago, two coordinated studies were started to determine the correlation between carcinogenic and mutagenic activity. Present data from these studies have clearly demonstrated the utility of some of the assays for genetic activity as short-term tests for mutagenicity which can be used for a meaningful toxicological evaluation of the numerous man-made chemicals in our environment. In both of these studies, large numbers of chemical carcinogens and non-carcinogenic structural analogs were tested for mutagenic activity. In these studies, which were started about the same time both in the United States and Japan, present data show that at least 80% of the chemical carcinogens are mutagens and less than 10% of the chemicals believed to be noncarcinogenic give false positive results. In the early 50's a similar study was made on the mutagenicity of chemical carcinogens and present data are clearly in conflict with the data collected at that time which showed little correlation between carcinogenic and mutagenic activity. These early experiments led to the premature demise ot~ the somatic mutation theory of cancer in the early 50's, and the general acceptance of these data made work in this area extremely difficult because of the low priority given to funding for new research in this area for m a n y years. Some investigators persisted in this line of research, however, and it has taken over t w e n t y years to develop a data base that shows quite clearly that the conclusion reached in the early 50's was wrong. It is important to remember that in the early 50's we knew nothing about the process of mutation, and we did not realize that certain strains of bacteria might detect only specific types of genetic alterations. We also did not appreciate the importance of metabolic activation -- and the fact that certain chemicals were n o t carcinogenic themselves but had to be activated by metabolism in various mammalian organs to a different form which actually possessed carcinogenic activity. The early tests were doomed to failure for the additional reason that these carcinogenically active metabolites were n o t evaluated for mutagenic activity, only the original chemicals were.
During the past ten years, very sensitive assays have been developed to detect the induction of particular types of genetic alterations. Other assay systems have been developed which are sensitive indicators of inhibition of activity of those enzymes responsible for DNA repair. We have also made remarkable progress in the development of in vitro techniques for metabolic activation so that we can produce in the test tube many of the same metabolites found in the whole mammal. A wide range of assay systems is being used in Japan and the United States to study mutagenic activity of chemical carcinogens. In a meeting of experimenters from b o t h countries in Honolulu held in December 1974, where the current status of these ongoing experiments was reviewed, it was apparent that the best correlation was obtained with the microbial assay systems for mutation-induction or DNA repair in Salmonella typhimurium, Escherichia coli and Bacillus subtilis in combination with tests for in vitro metabolic activation. The correlation was even better when the data from individual assays were combined. The studies were designed to compare the mutagenic activity of known chemical carcinogens and non-carcinogenic structural analogs. The assays showed remarkable sensitivity in that they were able, in the main part, to distinguish between the two; in other words, the carcinogenic chemicals gave positive results where the non-carcinogenic structural analogs gave negative results. In Japan a total of 60 c o m p o u n d s are under test and in the United States a total of 105, giving a total of 152 different compounds. In the assays for mutation induction, the best correlation is with Salmonella and at least 70--75% of the carcinogens tested with this assay show mutagenic activity. An even more recent tabulation of chemicals tested with the Salmonella assay system by Ames and his colleagues, using strains TA98 and TA100, shows that out of 139 chemical carcinogens tested at least 85% gave positive results. Furthermore, tests on an even larger number of non-carcinogenic chemicals selected at random shows that (only) a b o u t 1--2% are positive. This is an especially important point because when non-carcinogenic chemicals are tested which are not structural analogs of known carcinogens, the percentage of chemicals which show positive data appears to be very low. In other words, not all chemicals have genetic activity and the general impression that one gets when compounds are selected more or less at random is that the actual percentage of compounds showing positive results is very low. Nothing is known, of course, about the carcinogenic activity of the c o m p o u n d s showing positive results in such screening tests. Much more work is going to be required to determine whether those compounds which are positive actually possess carcinogenic activity. Obviously, they need to be tested more intensively for carcinogenic activity if, indeed, they have been tested at all. It is becoming obvious to everyone that what has been developed in this exploratory work is the capability to screen environmental chemicals for two important toxicological properties: (1) mutagenic activity and (2) carcinogenic activity. It is also important to emphasize that at this time they are not believed to be definitive tests b u t tests for presumptive mutagenic and carcinogenic activity in man and no more. There is no d o u b t that these tests properly used have the capability to assay large numbers of chemicals rapidly, efficiently and inexpensively to determine whether a given chemical or its metabolites have
13 genetic activity (and presumptive carcinogenic activity because of the high correlation between these t w o activities which I have already discussed). We visualize these short-term tests as being used in Tier 1 of a multi-tiered battery of tests which will enable us to establish priorities for testing in higher organisms. These latter tests will not only provide the quantitative data on a broader spectrum of genetic effects b u t will also enable us to develop a meaningful data base for benefit--risk evaluation. Development of this new approach for toxicological evaluation of environmental chemicals is b y no means complete; in fact, the work has barely started. This does n o t mean that we cannot exploit the progress that has already been made and put these tests to practical use. In other words, these exploratory experiments show a high enough correlation between carcinogenic and mutagenic activity and few enough false positives that the short-term tests will serve as a highly efficient prescreen. To further validate this approach, we need to test a larger number of known chemical carcinogens. In addition, careful attention must be paid to those carcinogens which give a negative test to determine whether they are related in structural functionality and might be detected as active b y a completely different assay. We have plans, for example, to begin a new program of testing to determine whether those carcinogens which are negative with regard to assays for mutation induction and DNA repair may give a positive test in assays for in vitro transformation. Perhaps the most comprehensive prescreen will require a combination of all three assay systems. This t y p e o f fine tuning of the short-term test to perfect the utilization as a prescreen will obviously take several years of additional developmental research. We are encouraged in this regard to achieve a beginning in the international coordination of this research and assignment of a high priority with regard to the required resources so that it will ensure rapid progress in this research area. This is true n o t only in the United States b u t also abroad and particularly in Japan. Because of the complexity of the task, we are also beginning to develop computer programs for data storage and retrieval to provide immediate access to such data for all interested parties. It will also make it possible to determine whether different investigators using the same assay systems get comparable results in tests on the same chemical as well as its metabolites. To achieve this goal we will require cooperation and coordination on an enormous scale. This approach seems to be the only practical solution to make it possible to compare objectively the relative utility of tests for mutation induction, DNA repair and in vitro transformation for use as prescreens in in vitro tests for carcinogenicity. It is also of interest that various industrial chemicals which are associated with occupational carcinogenesis have all been shown to be active in the short-term tests using Salmonella and in vitro metabolic activation by Ames and his colleagues. These include vinyl chloride, 4-amino-biphenyl, benzidine, 3,3'-dichlorobenzidine, 4-dimethyl-aminoazobenzene, beta-naphthylamine, 4nitrobiphenyl, N-nitro-dimethylamine, ~-propiolactone, bis(chloromethyl) ether and ethyleneimine. There is no d o u b t that a battery of short-term tests including assays for effects on DNA repair could be used on an even larger scale to try to identify potentially carcinogenic and mutagenic chemicals in widespread use in
industry. This would make it possible to take effective measures immediately to reduce exposure levels to ensure greater protection to our industrial workers. Such measures should help prevent or reduce the terrible burden in our society of occupational carcinogenesis. The short-term tests are simple enough so that they are not limited to tests on pure compounds. They can also be used to test commercial grade chemicals as well as the formulated products. This is an important concept in the current revision of our pesticide regulations, the toxic substance control act and other legislation to require more adequate testing of food additives and cosmetics. The tests are also simple enough so that they can be used easily for quality control to ensure that different lots of a given chemical produced by the same manufacturer or different manufacturer are biologically inactive. This is a particularly important concept because of the variation from lot to lot in commercial grade chemicals and the fact that t h e y can be contaminated with low levels of some other chemical with p o t e n t biological activity such as the contamination of herbicide 2,4,5-T with trace levels of dioxin. This capability will also make it possible to screen chemicals during early stages of product development by industry. The short-term tests provide a simple means of assuring with a high degree of probability that years of developmental work and tremendous expenditure of resources, often running into hundreds of thousands of dollars, will not be wasted with the discovery in the conventional animal bioassays performed at present that a valuable new product is carcinogenic. In most cases positive test data with the short-term tests should provide a sufficient data base to terminate plans for further development of a potential new product especially when genetically inactive structure analogs are also available. In the case of drugs, the genetic activity may be the basis for efficacy and further testing in higher organisms will be desirable to develop a more extensive data base for benefit-risk analysis. In some cases the benefit derived from treatment with a genetically active but unique life-saving drug may be worth the risk especially if the patient is beyond reproductive age -- or if the spectrum of genetic damage produced has no or a low probability of transmission. The biggest problem in the evaluation of the too numerous untested agents in our environment is how to establish priorities for testing. We have been fooled too often in the recent past to continue to classify compounds as active or inert ingredients based on intuition or badly designed experiments rather than on sound scientific data. The general public has been badly deluded with regard to the safety of hundreds of thousands of man-made chemicals to which they are constantly exposed. We have an illusion of safety, unfortunately based on the lack of meaningful tests, rather than on a thorough toxicological evaluation. We must make every effort as responsible scientists to make this illusion a reality through the development of an effective system of screening. During the past year, a great deal of progress has been made in developing a capability which can provide a mechanism for testing large numbers of untested environmental chemicals as well as their metabolites. There seems to be some question about the overall capability of this new methodology. I do not think we are, for example, ready to extrapolate directly from data obtained in short-
15 term tests for mutagenicity directly to man. In other words, I do not think we have a sufficiently large data base to really be confident that a genetically active c o m p o u n d in a short-term test will possess the same level of genetic activity in man. The short-term tests are best viewed as qualitative indicators of genetic activity. To get the quantitative data that are required for estimating risk to man, tests on other organisms will have to be performed. This is not only to obtain further information on the total spectrum of genetic alterations produced b u t also to determine the relative rate of increase over the spontaneous background. In that sense, I think it is important to stress again that the general consensus of opinion seems to be that these newly developed short-term tests provide a mechanism for alerting us to potential mutagenic and carcinogenic effects to man and that they are most effectively used at present to establish priorities for testing in higher organisms. In conclusion, I would like to return to a point that Dr. Auerbach made at the end of her prepared talk for this symposium, "We shall not ever be able to identify potential mutagens with complete confidence." A partial solution to this problem, of course, can be achieved by giving a higher priority to research aimed at making the short-term tests used in Tier 1 more comprehensive -- to eliminate to every extent possible false negatives and false positives. To eliminate false negatives perhaps a higher priority should be given to the development of assays for forward mutation which will detect any type of genetic alteration rather than reverse mutation which only detects particular types of genetic alterations. And perhaps a more thorough evaluation of the use of repair-deficient strains of various microorganisms should be made. We need to know to what extent any battery of tests used as a prescreen will yield false positives. By using the short-term tests to establish priorities for testing in higher organisms, we can develop a more extensive data base from tests on hundreds of compounds. It is only when we have established such a data base that we will really be able to determine the overall utility of the newly developed short-term tests -- and it is only then that we will be able to assign the short-term tests their proper role in the toxicological evaluation of the thousands of untested chemicals in our environment.