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Mutation Research, 38 ( 1 9 7 6 ) 1 6 5 - - 1 7 6 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
PROSPECTS F O R A R E V O L U T I O N IN THE METHODS OF TOXICOLOGICAL EVALUATION *
F R E D E R I C K J. DE S E R R E S
National Institute o f Environmental Health Sciences Research Triangle Park, North Carolina (U.S.A.) (Received October 25th, 1975) (Accepted November 28th, 1975)
Introduction During the past ten years we have witnessed the growth of an exciting new field in genetics, t hat of environmental mutagenesis. Interest in this field was generated by the realization t ha t there were m any man-made chemicals in the e n v i r o n m e n t with p o t e n t mutagenic activity in a wide range of experimental organisms. Because of these data there is now widespread concern over their effects on man [ 1 2 ] . A new area of research on these public health problems has emerged which is known as genetic toxicology. In contrast to ot her areas of toxicology, the focus in this new field is n o t only biological activity t hat may be harmful to the individual b u t also to his offspring. The responsibility of the genetic toxicologist is unusual in that first he must consider the effects of exposure to a genotoxic agent on individuals in the present population. Then, he must develop effective measures to determine the likelihood of successful transmission of any damage t h a t may be p r o duc e d in the germ cells and to evaluate its effect on future generations. This necessity not only to evaluate the effects of exposure on the individual but also to his offspring is technically difficult. Because man's most precious heritage is his genome, it is an awesome responsibility! The vitality of the h u m a n population and indeed its very existence, as we now know it, is dep e n d e n t in a large part on the successful transmission of an undamaged genome from one generation to another. We are now aware of the presence of agents in o u r en v ir o n men t which have the potential to interfere with this process. What we have witnessed during the past ten years is an ever-increasing incidence, in the literature, of papers on the mutagenic activity in experimental organisms of chemicals in widespread distribution. New data indicate t hat these chemicals m a y provide a n o t h e r hazard for man as well. * P r e s e n t e d at the Fifth Annual Meeting of the E u r o p e a n 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 y . F l o r e n c e , Italy. October 19--22, 1975.
166 During the past 18 months, new data have been collected that show a high correlation between carcinogenic and mutagenic activity. These new data show that at least 89% of chemical carcinogens are also mutagens. We are now concerned about the reverse correlation, that between mutagenic and carcinogenic activity. How many mutagens are also carcinogens? This is a question not only of great theoretical interest, but one of great practical importance to workers in this field of research. Many of us use potent mutagens in our own research programs. In our teaching laboratories there may also be exposure problems for young students of reproductive age. These are not abstractions, but problems that may impact heavily on the workers in the field of chemical mutagenesis as well as the students who will replace them. To be truthful, many research workers in this new field are concerned about the consequences of their own exposure to p o t e n t chemical mutagens in the past. Exploratory research in this new field has identified an important problem for the human population all over the world. It has also created a need for the development of agressive new research programs to develop highly efficient assay systems for mutagenic activity to screen untested environmental chemicals as well as to develop better methods to determine their effect on man. These needs have not been fully met, but the research in the area of screening techniques has progressed sufficiently, to put these newly developed assay systems to practical use in mass screening programs. This new technology will have an enormous impact on our society because of the capability that it has provided, for rapid, efficient and inexpensive toxicological evaluation. It is the development of this capability and its impact that I want to discuss in greater detail. Mutagens in our environment Concern over the general problem of environmental mutagenesis began with the discovery of supermutagens in the mid-sixties [31]. These are chemicals which can produce high frequencies of gene mutations at high levels of survival. In other words, high frequencies of mutations are produced at exposure levels which result either in low or negligible levels of toxicity to the cell. Great concern was expressed at the time, by many leading geneticists, over the possibility that such chemicals might be in widespread distribution [9]. Such a possibility could exist because such chemicals had either passed through traditional toxicological screening procedures or because they never were tested at all. The concern was amplified by the realization that because of the marked specificity of some supermutagens, man could be exposed to chemicals which could be potent inducers of gene mutations in the germ line, without exhibiting other types of more visible genetic damage, or cell-killing effects that would alert us to their presence. The exploratory experiments which were started in the late sixties to test chemicals in our environment, have shown that mutagenic chemicals can be found in all major categories: food additives, drugs, pesticides, cosmetics, air and water pollutants, as well as household and industrial chemicals. Some of the chemicals which were found to be mutagenic in these experiments can also be classified as super mutagens; ICR-170, AF-2, hycanthone, fi-propriolactone are good examples.
167 The discovery that there are many mutagenic chemicals in widespread distribution, with extensive human exposure in many cases, has created worldwide concern over their effects on man. Representative examples of the types of problems which have been encountered in this exploratory work will illustrate the magnitude of the task before us. Mutagenieity of food preservatives In 1973 Japanese scientists discovered that a chemical used widely as a food preservative in their country since 1965, a nitrofuran derivative called AF-2, was a potent mutagen in an E. coli [ 18,20] as well as in human cells in culture [35]. Other experiments with Salmonella [26], yeast and Neurospora [28] which were performed subsequently in this country rapidly confirmed the Japanese data. At a meeting which was held in Hakkone, Japan, early in August, 1974, these scientists, and others in the Japanese Ministry of Health, expressed grave concern over the effect of this exposure on the Japanese population. Their concern was based on the fact that AF-2 was used for ten years to preserve soybean curd, fish and meat sausage and many other foods considered as staples in the Japanese diet; so they were faced with a problem that affected essentially every living person in the Japanese population. In late August, 1974, Japanese scientists also found that AF-2 produced cancer of the forestomach in mice [17,36]. On the basis of all these data, the use of AF-2 as a food preservative was immediately banned. As a result of this ban, all AF-2 containing foods were removed from the market. It is well known that other food and feed additives in widespread use such as sodium nitrite [6,37], sodium bisulfite [16] and other nitrofuran derivatives structurally similar to AF-2 [24,34,39], are also mutagenic in experimental organisms. In the case of sodium nitrite and sodium bisulfite, which are used extensively to preserve many foods consumed by man, especially in the United States, the situation is even more complex since they are not as p o t e n t mutagens as AF-2. The problem is compounded by the fact that we have no way at present to evaluate the effects of chronic exposure to any type of mutagenic agent on man himself. Mutagenicity of pesticides Many pesticides in widespread use are also potent mutagens in experimental organisms. Good examples are ethylene oxide, captan [5], ethylene dibromide [11], heptachlor and chlordane to mention a few. The latter three, for example, have also been found recently to be carcinogenic in tests on rats and mice b y the National Cancer Institute. All of these chemicals have been extensively used in the past. We are probably only beginning to see the cancer in workers which will result from the high levels of occupational exposures that took place in these industries. Another chemical, which is used widely as a herbicide on most commercially grown corn, has been f o u n d to be converted to a p o t e n t mutagen. The studies of Gentile and Plewa [15] have shown that Atrazine, which is n o t mutagenic itself, is metabolized by the corn plant to a derivative that is a p o t e n t mutagen in
168 yeast. Atrazine is usually sprayed on the soil at the same time the corn kernels are planted. Assays for mutagenic activity on young corn plants showed that the plant sap contained a mutagenic derivative which caused reverse m ut at i on and gene conversion in two different strains of yeast. The mutagenic activity of plant sap e x t r a c t e d from mature corn plants or from the mature kernels, has not as y e t been determined. These data have alerted us to the possibility t hat a crop plant can metabolize a non-mutagenic pesticide to form what is a p o t e n t mutagen. In this c o u n t r y corn is one of our major crop plants. The corn plants themselves are used as siliage for dairy cows and ot her farm animals, and the ears of corn are used both as animal feed, as well as for m any products consumed by man. It will also be of interest to determine whether Atrazine has a mutagenic effect on the corn plant itself. This could create problems, if the appropriate measures are n o t taken, in the maintenance of the inbred strains used for breeding purposes. This i m p o r t a n t discovery points to the need for a much more extensive research program on this widely used herbicide. Mutagenicity o f drugs Many drugs have also been found to be mutagenic, one of the most extensively studied in recent years is h y c a n t h o n e (Etrenol), which is used to treat schistosomiasis (for review, see ref. [10] ). Schistosomiasis is a parasitic worm disease that is second in importance, only to malaria in the tropics. This disease affects millions of people. H y c a n t h o n e has been found to be especially effective by the World Health Organization in extensive field trials involving hundreds of thousands of patients. H y c a n t h o n e can also be classified as a supermutagen in experimental organisms [ 8 , 2 7 ] . The scanty epidemiological studies that have been performed, in connect i on with the field trials on this drug, are totally inadequate to evaluate its long-term genetic effects on treated populations. Many research workers fear that we may be trading off a t e m p o r a r y cure for schistosomiasis for some ot he r more serious disease. Genetic damage induced by h y c a n t h o n e in somatic cells of treated patients could lead to cancer in later years. Damage to germ cells could produce an increase in various genetic diseases which could persist for many generations. Obviously this is a complicated problem with complex benefit-risk implications.
Mutagenicity o f
cosmetics
In the field of cosmetics, scientists in the United Stated, England and Japan have f o u n d that the majority of commercial hair dyes available in those countries are p o t e n t mutagens in various short-term tests for mutagenicity. In the United States, the work in Dr. Bruce Ames' laboratory has shown [2] that 89% (150/169) of commercial oxidative-type hair dye preparations are mutagenic in Salmonella. At a conference held in Seattle in July, 1975, Dr. Takashi Sugimura, Director of the National Cancer Research Center, presented data from similar studies on the hair dyes available in Japan. His data using Salmonella, as well as repair-deficient strains of E. coli, B. subtilis and yeast have shown t hat 82% ( 1 4 6 /1 7 9 ) of Japanese hair dyes are mutagenic (T. Sugimura, pers. com-
169 mun.). Similar studies have been performed in England [32] which show that similar problems exist in commercially available hair dyes in that country. This problem is still under active investigation. Assays for the mutagenicJty of the 17 chemicals used in most commercial preparations are now being made with other experimental organisms. Basically this work is focused on determining which of these chemicals possess mutagenic activity. In addition, their potency is being evaluated with assay systems that can detect both genic and chromosomal damage. There are data from tests on human subjects that show that hair dyes are taken up through the scalp during the dyeing procedure [ 1 9 ] , and that dye ingredients can be found in the urine for some time after application [23]. Thus, there is a possibility of complete systemic exposure. To make the problem even more complex the addition of peroxide to the color base, prior to application, produces a myriad of analogs of similar and even more complex structure. In what form do they exist in the human b o d y ? What is the tissue distribution? H o w rapidly are they excreted? And in what form? Does the urine contain mutagenic metabolites? All are important additional questions that must be answered to evaluate the risks of using these hair dyes by the consumer. This finding of the mutagenic activity of commercial hair dyes raises the question as to whether other commercially available cosmetics might not also be genetically active. One might question the lack of a requirement for more rigorous toxicological evaluation of such products prior to their sale and distribution to the general public. It is estimated that in this country alone the population at risk from the use of hair dyes is 20,000,000 people. What evidence do we have that the dyes used in the manufacture of other cosmetics are any safer? Mutagenicity of industrial chemicals Numerous chemicals in widespread use in industry are also mutagenic in experimental organisms [25]. These include/3-propriolactone, ethyleneimine, 4amino-biphenyl, 4-nitro-biphenyl, bis(chloromethyl) ether, benzidine, and vinyl chloride, to mention a few. All of these have been associated with occupational carcinogenesis in the past few years. With the possible exception of vinyl chloride, the genetic effects of exposure to these chemicals has not been adequately evaluated, if indeed any tests have been made at all. Vinyl chloride was used in industry as a propellent in aerosol sprays, including hair sprays, household products and pesticide sprays. It is also polymerized to form polyvinyl chloride to make packaging materials, plastic containers for alcoholic beverages and many other products. Recently it has been shown that vinyl chloride has both carcinogenic and mutageni~ activity. Vinyl chloride has been shown to cause angiosarcoma of the liver in rats and this same disease has recently been found in vinyl chloride workers in four factories in the United States (for review, see ref. [4] ). It is of great interest that vinyl chloride has been found to give positive results in short-term tests for mutagenicity using either Salmonella [3,30], or yeast [21]. Recently it has been shown that vinyl chloride has produced signifi-
170 cant levels of chromosome damage in somatic cells of exposed workers [13, 14]. All of the other industrial chemicals have also been shown to give positive results in the Salmonella assay devised by Ames [25]. Had we had this information ten years ago, it is interesting to speculate that it might have been possible to reduce or eliminate the industrial carcinomas which are now being attributed to high exposure to these chemicals in the past. This exploratory work on chemicals in widespread use during the past six years has shown quite clearly that the concern expressed by geneticists in the late 60's was valid. There are many chemicals to which we are all exposed with mutagenic potential in experimental organisms. Specificity of chemical mutagens One thing that we have also learned about chemicals possessing mutagenic activity, in this exploratory work, that makes them especially difficult to detect reliably, is that they can produce highly specific types of genetic damage. This specificity is in sharp contrast to radiation which produces all types of damage. It is entirely possible, for example, for a chemical to produce abnormal numbers of chromosomes by causing nondisjunction during cell division but not gene mutations or chromosome aberrations. It is also possible for a chemical to produce gene mutations only by point mutation. This, a negative test with many assay systems, does not always mean that an agent is not mutagenic. The conclusion that we have drawn is that screening tests must include a battery of tester strains to make this assay comprehensive [12]. Tile primary concern over the effects of environmental mutagens on man is that exposure may produce damage in germ cells which will be transmitted to future generations. There is little doubt that this damage will produce higher frequencies of various types of disease which are known to have a genetic basis [12]. Unfortunately, with man we can only detect particular types of genetic damage in somatic cells rather than all types of damage in both somatic cells and germ cells. By withdrawing a blood sample we can culture peripheral lymphocytes and make an analysis for abnormal numbers of chromosomes or chromosome rearrangements. Unfortunately, a negative test would not rule out the possibility, that exposure to some agent did not produce a high frequency of gene mutations. Thus, if we use data derived from exposure of man himself, we may be grossly misled with regard to the actual hazard of exposure to a particular chemical. We have the illusion of safety, based on our inability to perform the appropriate test, rather than from sound scientific data! Development of short-term tests for mutagenicity To make tests of environmental chemicals with experimental organisms relevant for man, it is important not only to test the original chemical, but also the derivatives that may be formed as a result of mammalian metabolism. In experiments to develop in vitro techniques for metabolic activation Mailing [22] and Slater et al. [33] have shown that carcinogens could be activated in vitro by mouse liver homogenates. This m e t h o d has been put to more general use [1],
171 and more extensive tests on chemicals that require metabolic activation for carcinogenic activity have shown that many also possess mutagenic activity. This new approach provided a simple mechanism, to circumvent the requirement for testing chemicals in whole animals, and for utilizing assay systems which are more sensitive indicators of various types of genetic damage. Ames first showed t h a t the microsomal fraction could be incorporated into a thin layer of agar, along with the bacteria, in his spot test with Salmonella on Petri plates [1]. This same approach is now used by other investigators with various strains of bacteria and fungi [38]. The short-term tests for mutation-induction include assays for both forwardand reverse-mutation at specific loci. They also include tests for inhibition of DNA repair, by comparing the zones of inhibition obtained with wild-type and various repair-deficient mutants. The beauty of many of these new tests, is that it is possible to determine the mutagenic activity of a given chemical, over a wide range of concentrations, within a few days. These tests are rapid, sensitive and inexpensive and suitable for use in screening programs to evaluate the mutagenic activity of the large number of untested chemicals in our environment. The short-term tests are simple enough, so that testing need not be limited to the "active" ingredients of formulated products. They can be used to test all ingredients, both active and inert, as well as the formulated product itself. This is a marked departure from traditional toxicological evaluation which neglected to test those chemicals which were assumed to be inert. It is interesting to look back a few years and note that vinyl chloride was classified as an " i n e r t " ingredient in many aerosol sprays, in which it was used as a propellant. This new approach will provide greater safety to the consumer. The general utility of this new technology is the subject of great debate between scientists and legislators in the current revision of our pesticide regulations in the United States, the development of an effective Toxic Substances Control Act and other legislation to require more thorough testing of food additives and cosmetics. Mutagenicity of chemical
carcinogens
During the past two years these newly developed short-term tests have been used by investigators in the United States and Japan to study the correlation between carcinogenic and mutagenic activity. At a workshop which was held in Honolulu in December, 1974, to review the status of these ongoing experiments, it was apparent that the best correlation was obtained with the microbial assay systems. These were assays for mutation-induction or DNA repair in Salmonella typhimurium, Escherichia coli and Bacillus subtilis in combination with in vitro metabolic activation. The correlation was even better when the data from individual assays were combined [7]. The studies were designed to compare the mutagenic activity of known chemical carcinogens, non-carcinogenic structural analogs and other non-carcinogenic chemicals. The assays showed remarkable sensitivity in that they were able, in the main part, to distinguish between the first two classes. In other words, the carcinogenic chemicals gave positive mutagenicity test results whereas the non-carcinogenic structural analogs gave negative results. In those few cases where non-carcinogenic structural analogs were found to be mu-
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tagenic, there is strong suspicion that this may be an indication of inadequate carcinogenicity testing on laboratory animals. In the assays for mutation-induction, the best correlation between carcinogenic and mutagenic activity is with Salmonella; at least 70--75% of the carcinogens tested with this system show mutagenic activity. An even more recent tabulation of chemicals tested with the Salmonella assay system by Ames and his colleagues, using two new tester strains TA98 and TA100, shows that out of 175 chemical carcinogens tested 89% are mutagens (B.N. Ames, pers. commun.). Furthermore, tests on about 106 chemicals selected at random (including presumed non-carcinogenic structural analogs of known chemical carcinogens) show that only about 14% are mutagens (B.N. Ames, pers. commun.). Furthermore, when the chemicals structurally related to known carcinogens are eliminated, the percentage of chemicals which are mutagens is even lower (B.N. Ames, pers. commun.). As you might expect in such cases, the carcinogenic activity of the compounds giving a positive test for mutagenicity is not known. Obivously, these compounds need to be tested in longterm tests for carcinogenic activity in laboratory animals as well as in mutagenicity tests with other experimental organisms to determine the validity of this new approach. In this connection, mutagenic chemicals structurally related to known carcinogens should be tested for purity to ensure that the positive tests are not due to contamination of the sample being tested with a known mutagenic carcinogen. Implications for testing programs The basic question is then, whether these short-term tests which have been developed and validated in tests on several hundred chemicals, should be used on an even broader scale. With this approach, thousands of untested environmental chemicals could be assayed to try to identify those agents in our envir o n m e n t with potential mutagenic and carcinogenic affects on man. We are not ready, however, to extrapolate directly from data obtained in short-term tests for mutagenicity directly to man. This is simply because we do not have a sufficiently large data base to be confident that a genetically active c o m p o u n d in the short-term tests will produce significant levels of genetic damage in man. Because of the lack of such data 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, but also to determine the rate of increase over the spontaneous background for each class of genetic alteration. The general consensus is that these newly developed short-term tests provide a mechanism for alerting us to potential mutagenic and carcinogenic agents and that they are most effectively used at present to establish priorities for testing in higher organisms. There is concern that any battery of tests used as a prescreen may not be completely comprehensive and that some percentage of the chemicals tested will yield false negative and false positive results. Of particular concern are the false negatives, if no further testing is planned. To eliminate false negatives, a
173 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. To make the assay even more comprehensive perhaps a more thorough evaluation should be made of the use of repair-deficient strains of various microorganisms. A large number of such strains exist, but very few have been studied to determine their specificity and general utility as tester strains in mass screening programs. The addition of other strains as testers will be required to ensure that the short-term tests will detect any type of genetically active compound. We also need to know to what extent any battery of tests used as a prescreen will yield false positives. These are chemicals which will give positive results in the short-term test but not in higher organisms. 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 be able to determine the efficacy of the short-term tests and to validate this new approach. Future course The committee 17 Report of the Environmental Mutagen Society on Enviromental Mutagenic Hazards which appeared in the February 14 issue of Science this year recommended strongly that "screening should be initiated as rapidly and as extensively as possible." As a result of the development of the short-term tests, we now have a means for rapid, comprehensive and inexpensive screening. We have been given an unprecedented opportunity to revolutionize traditional methods of toxicological evaluation and to eliminate disease-causing agents from our environment. This approach should enable us to prevent an increase in the frequency of various diseases known to have a genetic basis, as well as to reduce the frequency of cancers resulting from environmental and occupational exposure. The shortterm tests are not perfect, but no test system ever developed provides 100% ascertainment. With time we can undoubtedly perfect this approach so that the activity of chemicals which give a false negative test with present techniques will also be detected. The general utilization of this new technology will enable us to evaluate numerous untested chemicals in our environment and to evaluate their mutagenic and carcinogenic potential for man. The data from exploratory studies with these newly developed short-term tests clearly "indicate that we have a problem of great concern. The challenge presented to geneticists by the recognition of this problem can only be met effectively by the development of new theoretical and applied research programs. We not only need assays with a higher level of ascertainment that will n o t miss mutagenically active agents, but we also need better ways to evaluate the risk of exposure to active agents for man. Finally, we need better test methods that can be used on man himself to determine the frequency and type of damage produced by exposure to an active agent. This is a situation that will require great ingenuity, creativity and imagination to resolve these difficult problems. Our success may not only have a marked influence on the quality of life, but because we are concerned about effects that are transmitted from one genera-
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tion to another and which are a c c u m u l a t e d with time, our success m a y well determine the future and very existence o f man on this planet. Who a m o n g us can afford to ignore such a challenge?
References 1 A m e s , B.N., W.F. D u r s t o n , E. Y a m a s a k i a n d F.D. Lee, C a r c i n o g e n s are m u t a g e n s : a s i m p l e test s y s t e m c o m b i n i n g liver h o m o g e n a t e s for a c t i v a t i o n a n d b a c t e r i a for d e t e c t i o n , Proc. Natl. Acad. Sci. ( U S ) . , 70 (1973) 2281--2285. 2 A m e s , B.N., H . O . K a m m e n a n d E. Y a m a s a k i , Hair d y e s are m u t a g e n i c : i d e n t i f i c a t i o n of a v a r i e t y of m u t a g e n i c i n g r e d i e n t s , Proc. Natl. A c a d . Sci. (US)., 72, ( 1 9 7 5 ) 2 4 2 3 - - 2 4 2 7 . 3 B a r t c h , H., C. Malaveille a n d R. M o n t e s a n o , H u m a n , rat a n d m o u s e l i v e r - m e d i a t e d m u t a g e n i c i t y o f v i n y l c h l o r i d e in S. l y p h i m u u i u m strains, Int. J. C a n c e r , 15, No. 3 ( 1 9 7 5 ) , 4 2 9 - - 4 3 7 . 4 B a r t c h , H. a n d R. M o n t e s a n o , M u t a g e n i c a n d c a r c i n o g e n i c e f f e c t s of v i n y l c h l o r i d e , M u t a t i o n Res., 32 (1975) 93--114. 5 Bridges, B.A., T h e m u t a g e n i c i t y of c a p t a n s a n d r e l a t e d fungicides, M u t a t i o n Res., 32 ( 1 9 7 5 ) 3 - - 3 4 . 6 B r o c k m a n , H . E . , F.J. de Serres a n d W.E. B a r n e t t , Analysis of a d - 3 m u t a t i o n s i n d u c e d by n i t r o u s acid in a h e t e r o k a r y o n of N e u r o s p o r a cr¢*.ssa, M u t a t i o n Res., 7 ( 1 9 6 9 ) 3 0 7 - - 3 1 4 . 7 Bronkes, P. a n d F.J. de Serres, R e p o r t of the w o r k s h o p o n the m u t a g e n i e i t y of c h e m i c a l c a r c i n o g e n s , D e c e m b e r 9 - - 1 1 , 1 9 7 4 , H o n o l u l u , U.S.A., M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 5 5 - - 1 6 0 . 8 Clive, D., M u t a g e n i c i t y of t h i o x a n t h e n e s ( H y c a n t h o n e , L u c a n t h o n e , a n d f o u r I n d a z o l e d e r i v a t i v e s ) at the T K l o c u s in c u l t u r e d m a m m a l i a n cells, M u t a t i o n Res., 26 ( 1 9 7 4 ) 3 0 7 - - 3 1 8 . 9 C r o w , J . F . , C h e m i c a l risk to f u t u r e g e n e r a t i o n s , Scientist a n d Citizen, 10 ( 1 9 6 8 ) 1 1 3 - - 1 1 7 . 10 De Serres, F.J. cd., L o n g - t e r m t o x i c i t y of a n t i s c h i s t o s o m a l drugs., J. T o x . E n v i r o n . H l t h , 1 ( 1 9 7 5 ) . 11 De Serres, F.J. a n d H.V. Malling, G e n e t i c analysis of a d - 3 m u t a n t s of N e u r o s p o r a crassa i n d u c e d b y e t h y l e n e d i b r o m i d e - - A c o m m o n l y used p e s t i c i d e , EMS N e w s l e t t e r , No. 4 ( 1 9 7 1 ) 3 6 - - 3 7 . 12 D r a k e , J.W., S. A b r a h a m s o n , J . F . C r o w , A. H o l l a e n d e r , S. L e d e r b e r g , M.S. L e g a t o r , J.V. Neel, M.W. S h a w , H.E. S u t t o n , R.C. y o n Borstel a n d S. Z i m m e r i n g , E n v i r o n m e n t a l m u t a g e n i c h a z a r d s , Science, 187 ( 1 9 7 5 ) 5 0 3 - - 5 1 4 . 13 D u c a t m a n , A., K. H i r s c h h o r n a n d l.J. Selikoff, V i n y l chloride a n d h u m a n c h r o m o s o m e a b e r r a t i o n s , M u t a t i o n Res., 31 ( 1 9 7 5 ) 1 6 3 - - 1 6 8 . 14 F u n e s - C r a v i o t o , F., B. L a m b e r t , J. L i n d s t e n , L. E h r e n b e r g , A . T . N a t a r a j a n a n d S. O s t e r m a n - G o l k a x s , C h r o m o s o m e a b e r r a t i o n s in w o r k e r s e x p o s e d to vinyl c h l o r i d e , L a n c e t , I, No. 7 9 0 4 ( 1 9 7 5 ) 4 5 9 . 15 Gentile, J.M. a n d M.J. Plewa, A bioassay for s c r e e n i n g h o s t - m e d i a t e d p r o x i m a l m u t a g e n s in agricult u r e , M u t a t i o n Res., 31 ( 1 9 7 5 ) 319. 16 Iida, S., M. l n o u e , K. Kai, N. K i t a m u r a , I. K u d o , M. S o n o , T. T s u r v o , H. H a y a t s o , A. Miura a n d Y. W a t a y a , S o m e p r o p e r t i e s of the d a m a g e of D N A a n d p h a g e l a m b d a i n d u c e d b y bisulfite, M u t a t i o n Res., 26 ( 1 9 7 4 ) 4 3 3 - - 4 3 4 . 17 I k e d a , Y., S. H o r i u c h i , T. F u r u y a , O. U c h i d a , K. Suzuki a n d J. A z e g a m i , I n t e r i m R e p o r t : I n d u c t i o n o f Gastric T u m o r s in Mice by F e e d i n g o f F u r y l f u r a m i d e , F o o d S a n i t a t i o n S t u d y Council, Minister of H e a l t h a n d Welfare, J a p a n , A u g u s t ( 1 9 7 4 ) . 18 K a d a , T., E s c h e r i c h i a coli m u t a g e n i c i t y of f u r y l f o r a m i d e , J a p a n . J, G e n e t . , 48 ( 1 9 7 3 ) 3 0 1 - - 3 0 5 . 19 Kiese, M. a n d E. R a u s c h e r , T h e a b s o r p t i o n of p - t o l u e n e d i a m i n e t h r o u g h h u m a n skin in hair d y e i n g , T o x i c . A p p l . P h a r m a c . , 13 ( 1 9 6 8 ) 3 2 5 - - 3 3 1 . 20 K o n d o , S. a n d H. I c h i k a w a - R y o , T e s t i n g a n d classification o f m u t a g e n i c i t y of f u r y l f u r a m i d e in E s c h c r i c h i o coli, J a p a n . J. G e n e t . , 48 ( 1 9 7 3 ) 2 9 5 - - 3 0 0 . 21 L o p r i e n o , N., R. Barale, S. Baroncelli, C. Bauer, G. B r o n z e t t i , A. C a m m e l l i n i , G. Cercignani, C. Corsi, G. Gervasi, C. L e p o r i n i , R. Nieri, M. Rossi, G. S t r e t t i a n d G. T u r e h i , E v a l u a t i o n of the g e n e t i c e f f e c t s of v i n y l c h l o r i d e m o n o m e r (VCM) u n d e r the i n f l u e n c e of liver m i c r o s o m e s (in press). 22 Mailing, H . V . , D i m e t h y l n i t r o s a m i n e : f o r m a t i o n of m u t a g e n i c c o m p o u n d s b y i n t e r a c t i o n w i t h m o u s e liver m i c r o s o m c s , M u t a t i o n Res., 13 ( 1 9 7 1 ) 4 2 5 - - 4 2 9 . 23 Marshall, S. a n d W.S. P a l m e r , D a r k u r i n e a f t e r hair c o l o r i n g , J. A m e r . Med. Ass., 2 2 6 ( 1 9 7 3 ) 1 0 1 0 . 24 McCalla, D.R. a n d D. V o u t s i n o s , On the m u t a g e n i c i t y of n i t r o f u r a n s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 3 - - 1 6 . 25 M c C a n n , J. a n d B.N. A m e s , A s i m p l e r m e t h o d for d e t e c t i n g e n v i r o n m e n t a l c a r c i n o g e n s as m u t a g e n s , A n n . N.Y. A c a d . Sci., 2 6 9 ( 1 9 7 5 ) 2 1 - - 2 5 . 26 M c C a n n , J., N.E, S p i n g a r n , J. K o b o r i a n d B.N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s : b a c t e r i a l t e s t e r strains w i t h R f a c t o r plasmids, Proc. Natl. A e a d . Sei. ( U S ) , 72 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 . 27 Ong, T. a n d F.J. de Serres, M u t a g e n i c e v a l u a t i o n of a n t i s c h i s t o s o m a l a g e n t s in N e u r o s p o r a crassa, J. T o x . E n v i r o n m . H l t h , 1 ( 1 9 7 5 ) 291, 28 Ong, T.M. a n d S h a h i n , M.M., M u t a g e n i c a n d r e c o m b i n o g e n i c activities o f t h e f o o d a d d i t i v e f u r y l f u r a m i d e in e u k a r y o t e s , Science, 1 8 4 ( 1 9 7 4 ) 1 0 8 6 - - 1 0 8 7 .
175 2 9 P u r c h a s e , I . F . H . , C . R . R i c h a r d s o n , D. A n d e r s o n , C h r o m o s o m a l a n d d o m i n a n t l e t h a l e f f e c t s o f v i n y l c h l o r i d e , T h e L a n c e t , II, N o . 7 9 3 1 ( 1 9 7 5 ) 4 1 0 - - 4 1 1 . 3 0 R a n n u g , V., A. J o h a n s s o n , C. R a m e l a n d C . A . W a c h t m e i s t e r , Tile m u t a g e n i c i t y o f v i n y l c h l o r i d e a f t e r metabolic activation, Ambio, 3 (1974) 194--197. 31 R a p o p o r t , I . A . , N . N . Z o z , S.I. M a k a r o v a a n d T . V . S a l n i k o v a , S u p e r m u t a g e n s ( N a u k a , M o s c o w , 1 9 6 6 ) . 3 2 Searle, C.E., D . G . H a r n d e n , S. V e n i t t a n d O . H . B . G y d e , C a r c i n o g e n i c i t y a n d m u t a g e n i c i t y t e s t s o f some hair colourants and constituents, Nature, 255 (1975) 506--507. 3 3 S l a t e r , E . E . , M.D. A n d e r s o n a n d H.S. R o s e n k r a n z , R a p i d d e t e c t i o n of m u t a g e n s a n d c a r c i n o g e n s , C a n c e r R e s . , 31 ( 1 9 7 1 ) 9 7 0 - - 9 7 3 . 3 4 T a z i m a , Y., T. K a d a a n d A . M u r a k a m i , M u t a g e n i c i t y o f n i t r o f u r a n d e r i v a t i v e s , i n c l u d i n g f u r y l furamide, a food preservative, Mutation Res., 32 (1975) 55--80. 3 5 T o n o m u r a , A . a n d M.S. Sasaki, C h r o m o s o m e a b e r r a t i o n s a n d D N A r e p a i r s y n t h e s i s in c u l t u r e d h u m a n cells e x p o s e d t o n i t r o f u r a n s , J a p a n . J. G e n e t . , 4 8 ( 1 9 7 3 ) 2 9 1 - - 2 9 4 . 3 6 U c h i d a , O., T. F u r u y a , K. K a w a m a t a , S. H o r i c u c h i , Y. I k e d a , K . S u z u k i a n d J. AT.egami, S t u d i e s o n t h e t o x i c i t y o f f u r y l f u r a m i d e ( A F - 2 ) . II. S t u d y o n the c a r c i n o g e n i c i t y in m i c e , J a p a n . J. P h a r m a c . (in press). 3 7 V a l e n c i a , R., S. A b r a h a m s o n , P. W a g o n e r a n d L. M a n s f i e l d , T e s t i n g f o r f o o d a d d i t i v e - i n d u c e d m u t a t i o n s in Drosophila melano¢aster, M u t a t i o n R e s . , 21 ( 1 9 7 3 ) 2 4 0 - - 2 4 1 . 3 8 W e e k e s , V., J. Williams, P. B u t l e r , G . R o y a n d D. B r u s i c k , R a p i d s c r e e n i n g t e c h n i q u e s f o r m u t a g e n icity e v a l u a t i o n u s i n g m i c r o b i a l cells c o m b i n e d w i t h m a m m a l i a n a c t i v a t i o n f r a c t i o n s , M u t a t i o n Res. (in press). 3 9 Y a h a g i , T., M. N a g a o , K. H a r a , T. M a t s u s h i m a , T. S u g i m u r a a n d G . T . B r y a n , R e l a t i o n s h i p s b e t w e e n t h e c a r c i n o g e n i c a n d m u t a g e n i c o r D N A - m o d i f y i n g e f f e c t s o f n i t r o f u r a n d e r i v a t i v e s i n c l u d i n g 2-(2furyl)-3-(5-nitro-2-furyl) acrylamide, a food additive, Cancer Res., 34 (1974) 2266--2273.
Mutation Research, 38 ( 1 9 7 6 ) 1 6 5 - - 1 7 6 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
PROSPECTS F O R A R E V O L U T I O N IN THE METHODS OF TOXICOLOGICAL EVALUATION *
F R E D E R I C K J. DE S E R R E S
National Institute o f Environmental Health Sciences Research Triangle Park, North Carolina (U.S.A.) (Received October 25th, 1975) (Accepted November 28th, 1975)
Introduction During the past ten years we have witnessed the growth of an exciting new field in genetics, t hat of environmental mutagenesis. Interest in this field was generated by the realization t ha t there were m any man-made chemicals in the e n v i r o n m e n t with p o t e n t mutagenic activity in a wide range of experimental organisms. Because of these data there is now widespread concern over their effects on man [ 1 2 ] . A new area of research on these public health problems has emerged which is known as genetic toxicology. In contrast to ot her areas of toxicology, the focus in this new field is n o t only biological activity t hat may be harmful to the individual b u t also to his offspring. The responsibility of the genetic toxicologist is unusual in that first he must consider the effects of exposure to a genotoxic agent on individuals in the present population. Then, he must develop effective measures to determine the likelihood of successful transmission of any damage t h a t may be p r o duc e d in the germ cells and to evaluate its effect on future generations. This necessity not only to evaluate the effects of exposure on the individual but also to his offspring is technically difficult. Because man's most precious heritage is his genome, it is an awesome responsibility! The vitality of the h u m a n population and indeed its very existence, as we now know it, is dep e n d e n t in a large part on the successful transmission of an undamaged genome from one generation to another. We are now aware of the presence of agents in o u r en v ir o n men t which have the potential to interfere with this process. What we have witnessed during the past ten years is an ever-increasing incidence, in the literature, of papers on the mutagenic activity in experimental organisms of chemicals in widespread distribution. New data indicate t hat these chemicals m a y provide a n o t h e r hazard for man as well. * P r e s e n t e d at the Fifth Annual Meeting of the E u r o p e a n 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 y . F l o r e n c e , Italy. October 19--22, 1975.
166 During the past 18 months, new data have been collected that show a high correlation between carcinogenic and mutagenic activity. These new data show that at least 89% of chemical carcinogens are also mutagens. We are now concerned about the reverse correlation, that between mutagenic and carcinogenic activity. How many mutagens are also carcinogens? This is a question not only of great theoretical interest, but one of great practical importance to workers in this field of research. Many of us use potent mutagens in our own research programs. In our teaching laboratories there may also be exposure problems for young students of reproductive age. These are not abstractions, but problems that may impact heavily on the workers in the field of chemical mutagenesis as well as the students who will replace them. To be truthful, many research workers in this new field are concerned about the consequences of their own exposure to p o t e n t chemical mutagens in the past. Exploratory research in this new field has identified an important problem for the human population all over the world. It has also created a need for the development of agressive new research programs to develop highly efficient assay systems for mutagenic activity to screen untested environmental chemicals as well as to develop better methods to determine their effect on man. These needs have not been fully met, but the research in the area of screening techniques has progressed sufficiently, to put these newly developed assay systems to practical use in mass screening programs. This new technology will have an enormous impact on our society because of the capability that it has provided, for rapid, efficient and inexpensive toxicological evaluation. It is the development of this capability and its impact that I want to discuss in greater detail. Mutagens in our environment Concern over the general problem of environmental mutagenesis began with the discovery of supermutagens in the mid-sixties [31]. These are chemicals which can produce high frequencies of gene mutations at high levels of survival. In other words, high frequencies of mutations are produced at exposure levels which result either in low or negligible levels of toxicity to the cell. Great concern was expressed at the time, by many leading geneticists, over the possibility that such chemicals might be in widespread distribution [9]. Such a possibility could exist because such chemicals had either passed through traditional toxicological screening procedures or because they never were tested at all. The concern was amplified by the realization that because of the marked specificity of some supermutagens, man could be exposed to chemicals which could be potent inducers of gene mutations in the germ line, without exhibiting other types of more visible genetic damage, or cell-killing effects that would alert us to their presence. The exploratory experiments which were started in the late sixties to test chemicals in our environment, have shown that mutagenic chemicals can be found in all major categories: food additives, drugs, pesticides, cosmetics, air and water pollutants, as well as household and industrial chemicals. Some of the chemicals which were found to be mutagenic in these experiments can also be classified as super mutagens; ICR-170, AF-2, hycanthone, fi-propriolactone are good examples.
167 The discovery that there are many mutagenic chemicals in widespread distribution, with extensive human exposure in many cases, has created worldwide concern over their effects on man. Representative examples of the types of problems which have been encountered in this exploratory work will illustrate the magnitude of the task before us. Mutagenieity of food preservatives In 1973 Japanese scientists discovered that a chemical used widely as a food preservative in their country since 1965, a nitrofuran derivative called AF-2, was a potent mutagen in an E. coli [ 18,20] as well as in human cells in culture [35]. Other experiments with Salmonella [26], yeast and Neurospora [28] which were performed subsequently in this country rapidly confirmed the Japanese data. At a meeting which was held in Hakkone, Japan, early in August, 1974, these scientists, and others in the Japanese Ministry of Health, expressed grave concern over the effect of this exposure on the Japanese population. Their concern was based on the fact that AF-2 was used for ten years to preserve soybean curd, fish and meat sausage and many other foods considered as staples in the Japanese diet; so they were faced with a problem that affected essentially every living person in the Japanese population. In late August, 1974, Japanese scientists also found that AF-2 produced cancer of the forestomach in mice [17,36]. On the basis of all these data, the use of AF-2 as a food preservative was immediately banned. As a result of this ban, all AF-2 containing foods were removed from the market. It is well known that other food and feed additives in widespread use such as sodium nitrite [6,37], sodium bisulfite [16] and other nitrofuran derivatives structurally similar to AF-2 [24,34,39], are also mutagenic in experimental organisms. In the case of sodium nitrite and sodium bisulfite, which are used extensively to preserve many foods consumed by man, especially in the United States, the situation is even more complex since they are not as p o t e n t mutagens as AF-2. The problem is compounded by the fact that we have no way at present to evaluate the effects of chronic exposure to any type of mutagenic agent on man himself. Mutagenicity of pesticides Many pesticides in widespread use are also potent mutagens in experimental organisms. Good examples are ethylene oxide, captan [5], ethylene dibromide [11], heptachlor and chlordane to mention a few. The latter three, for example, have also been found recently to be carcinogenic in tests on rats and mice b y the National Cancer Institute. All of these chemicals have been extensively used in the past. We are probably only beginning to see the cancer in workers which will result from the high levels of occupational exposures that took place in these industries. Another chemical, which is used widely as a herbicide on most commercially grown corn, has been f o u n d to be converted to a p o t e n t mutagen. The studies of Gentile and Plewa [15] have shown that Atrazine, which is n o t mutagenic itself, is metabolized by the corn plant to a derivative that is a p o t e n t mutagen in
168 yeast. Atrazine is usually sprayed on the soil at the same time the corn kernels are planted. Assays for mutagenic activity on young corn plants showed that the plant sap contained a mutagenic derivative which caused reverse m ut at i on and gene conversion in two different strains of yeast. The mutagenic activity of plant sap e x t r a c t e d from mature corn plants or from the mature kernels, has not as y e t been determined. These data have alerted us to the possibility t hat a crop plant can metabolize a non-mutagenic pesticide to form what is a p o t e n t mutagen. In this c o u n t r y corn is one of our major crop plants. The corn plants themselves are used as siliage for dairy cows and ot her farm animals, and the ears of corn are used both as animal feed, as well as for m any products consumed by man. It will also be of interest to determine whether Atrazine has a mutagenic effect on the corn plant itself. This could create problems, if the appropriate measures are n o t taken, in the maintenance of the inbred strains used for breeding purposes. This i m p o r t a n t discovery points to the need for a much more extensive research program on this widely used herbicide. Mutagenicity o f drugs Many drugs have also been found to be mutagenic, one of the most extensively studied in recent years is h y c a n t h o n e (Etrenol), which is used to treat schistosomiasis (for review, see ref. [10] ). Schistosomiasis is a parasitic worm disease that is second in importance, only to malaria in the tropics. This disease affects millions of people. H y c a n t h o n e has been found to be especially effective by the World Health Organization in extensive field trials involving hundreds of thousands of patients. H y c a n t h o n e can also be classified as a supermutagen in experimental organisms [ 8 , 2 7 ] . The scanty epidemiological studies that have been performed, in connect i on with the field trials on this drug, are totally inadequate to evaluate its long-term genetic effects on treated populations. Many research workers fear that we may be trading off a t e m p o r a r y cure for schistosomiasis for some ot he r more serious disease. Genetic damage induced by h y c a n t h o n e in somatic cells of treated patients could lead to cancer in later years. Damage to germ cells could produce an increase in various genetic diseases which could persist for many generations. Obviously this is a complicated problem with complex benefit-risk implications.
Mutagenicity o f
cosmetics
In the field of cosmetics, scientists in the United Stated, England and Japan have f o u n d that the majority of commercial hair dyes available in those countries are p o t e n t mutagens in various short-term tests for mutagenicity. In the United States, the work in Dr. Bruce Ames' laboratory has shown [2] that 89% (150/169) of commercial oxidative-type hair dye preparations are mutagenic in Salmonella. At a conference held in Seattle in July, 1975, Dr. Takashi Sugimura, Director of the National Cancer Research Center, presented data from similar studies on the hair dyes available in Japan. His data using Salmonella, as well as repair-deficient strains of E. coli, B. subtilis and yeast have shown t hat 82% ( 1 4 6 /1 7 9 ) of Japanese hair dyes are mutagenic (T. Sugimura, pers. com-
169 mun.). Similar studies have been performed in England [32] which show that similar problems exist in commercially available hair dyes in that country. This problem is still under active investigation. Assays for the mutagenicJty of the 17 chemicals used in most commercial preparations are now being made with other experimental organisms. Basically this work is focused on determining which of these chemicals possess mutagenic activity. In addition, their potency is being evaluated with assay systems that can detect both genic and chromosomal damage. There are data from tests on human subjects that show that hair dyes are taken up through the scalp during the dyeing procedure [ 1 9 ] , and that dye ingredients can be found in the urine for some time after application [23]. Thus, there is a possibility of complete systemic exposure. To make the problem even more complex the addition of peroxide to the color base, prior to application, produces a myriad of analogs of similar and even more complex structure. In what form do they exist in the human b o d y ? What is the tissue distribution? H o w rapidly are they excreted? And in what form? Does the urine contain mutagenic metabolites? All are important additional questions that must be answered to evaluate the risks of using these hair dyes by the consumer. This finding of the mutagenic activity of commercial hair dyes raises the question as to whether other commercially available cosmetics might not also be genetically active. One might question the lack of a requirement for more rigorous toxicological evaluation of such products prior to their sale and distribution to the general public. It is estimated that in this country alone the population at risk from the use of hair dyes is 20,000,000 people. What evidence do we have that the dyes used in the manufacture of other cosmetics are any safer? Mutagenicity of industrial chemicals Numerous chemicals in widespread use in industry are also mutagenic in experimental organisms [25]. These include/3-propriolactone, ethyleneimine, 4amino-biphenyl, 4-nitro-biphenyl, bis(chloromethyl) ether, benzidine, and vinyl chloride, to mention a few. All of these have been associated with occupational carcinogenesis in the past few years. With the possible exception of vinyl chloride, the genetic effects of exposure to these chemicals has not been adequately evaluated, if indeed any tests have been made at all. Vinyl chloride was used in industry as a propellent in aerosol sprays, including hair sprays, household products and pesticide sprays. It is also polymerized to form polyvinyl chloride to make packaging materials, plastic containers for alcoholic beverages and many other products. Recently it has been shown that vinyl chloride has both carcinogenic and mutageni~ activity. Vinyl chloride has been shown to cause angiosarcoma of the liver in rats and this same disease has recently been found in vinyl chloride workers in four factories in the United States (for review, see ref. [4] ). It is of great interest that vinyl chloride has been found to give positive results in short-term tests for mutagenicity using either Salmonella [3,30], or yeast [21]. Recently it has been shown that vinyl chloride has produced signifi-
170 cant levels of chromosome damage in somatic cells of exposed workers [13, 14]. All of the other industrial chemicals have also been shown to give positive results in the Salmonella assay devised by Ames [25]. Had we had this information ten years ago, it is interesting to speculate that it might have been possible to reduce or eliminate the industrial carcinomas which are now being attributed to high exposure to these chemicals in the past. This exploratory work on chemicals in widespread use during the past six years has shown quite clearly that the concern expressed by geneticists in the late 60's was valid. There are many chemicals to which we are all exposed with mutagenic potential in experimental organisms. Specificity of chemical mutagens One thing that we have also learned about chemicals possessing mutagenic activity, in this exploratory work, that makes them especially difficult to detect reliably, is that they can produce highly specific types of genetic damage. This specificity is in sharp contrast to radiation which produces all types of damage. It is entirely possible, for example, for a chemical to produce abnormal numbers of chromosomes by causing nondisjunction during cell division but not gene mutations or chromosome aberrations. It is also possible for a chemical to produce gene mutations only by point mutation. This, a negative test with many assay systems, does not always mean that an agent is not mutagenic. The conclusion that we have drawn is that screening tests must include a battery of tester strains to make this assay comprehensive [12]. Tile primary concern over the effects of environmental mutagens on man is that exposure may produce damage in germ cells which will be transmitted to future generations. There is little doubt that this damage will produce higher frequencies of various types of disease which are known to have a genetic basis [12]. Unfortunately, with man we can only detect particular types of genetic damage in somatic cells rather than all types of damage in both somatic cells and germ cells. By withdrawing a blood sample we can culture peripheral lymphocytes and make an analysis for abnormal numbers of chromosomes or chromosome rearrangements. Unfortunately, a negative test would not rule out the possibility, that exposure to some agent did not produce a high frequency of gene mutations. Thus, if we use data derived from exposure of man himself, we may be grossly misled with regard to the actual hazard of exposure to a particular chemical. We have the illusion of safety, based on our inability to perform the appropriate test, rather than from sound scientific data! Development of short-term tests for mutagenicity To make tests of environmental chemicals with experimental organisms relevant for man, it is important not only to test the original chemical, but also the derivatives that may be formed as a result of mammalian metabolism. In experiments to develop in vitro techniques for metabolic activation Mailing [22] and Slater et al. [33] have shown that carcinogens could be activated in vitro by mouse liver homogenates. This m e t h o d has been put to more general use [1],
171 and more extensive tests on chemicals that require metabolic activation for carcinogenic activity have shown that many also possess mutagenic activity. This new approach provided a simple mechanism, to circumvent the requirement for testing chemicals in whole animals, and for utilizing assay systems which are more sensitive indicators of various types of genetic damage. Ames first showed t h a t the microsomal fraction could be incorporated into a thin layer of agar, along with the bacteria, in his spot test with Salmonella on Petri plates [1]. This same approach is now used by other investigators with various strains of bacteria and fungi [38]. The short-term tests for mutation-induction include assays for both forwardand reverse-mutation at specific loci. They also include tests for inhibition of DNA repair, by comparing the zones of inhibition obtained with wild-type and various repair-deficient mutants. The beauty of many of these new tests, is that it is possible to determine the mutagenic activity of a given chemical, over a wide range of concentrations, within a few days. These tests are rapid, sensitive and inexpensive and suitable for use in screening programs to evaluate the mutagenic activity of the large number of untested chemicals in our environment. The short-term tests are simple enough, so that testing need not be limited to the "active" ingredients of formulated products. They can be used to test all ingredients, both active and inert, as well as the formulated product itself. This is a marked departure from traditional toxicological evaluation which neglected to test those chemicals which were assumed to be inert. It is interesting to look back a few years and note that vinyl chloride was classified as an " i n e r t " ingredient in many aerosol sprays, in which it was used as a propellant. This new approach will provide greater safety to the consumer. The general utility of this new technology is the subject of great debate between scientists and legislators in the current revision of our pesticide regulations in the United States, the development of an effective Toxic Substances Control Act and other legislation to require more thorough testing of food additives and cosmetics. Mutagenicity of chemical
carcinogens
During the past two years these newly developed short-term tests have been used by investigators in the United States and Japan to study the correlation between carcinogenic and mutagenic activity. At a workshop which was held in Honolulu in December, 1974, to review the status of these ongoing experiments, it was apparent that the best correlation was obtained with the microbial assay systems. These were assays for mutation-induction or DNA repair in Salmonella typhimurium, Escherichia coli and Bacillus subtilis in combination with in vitro metabolic activation. The correlation was even better when the data from individual assays were combined [7]. The studies were designed to compare the mutagenic activity of known chemical carcinogens, non-carcinogenic structural analogs and other non-carcinogenic chemicals. The assays showed remarkable sensitivity in that they were able, in the main part, to distinguish between the first two classes. In other words, the carcinogenic chemicals gave positive mutagenicity test results whereas the non-carcinogenic structural analogs gave negative results. In those few cases where non-carcinogenic structural analogs were found to be mu-
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tagenic, there is strong suspicion that this may be an indication of inadequate carcinogenicity testing on laboratory animals. In the assays for mutation-induction, the best correlation between carcinogenic and mutagenic activity is with Salmonella; at least 70--75% of the carcinogens tested with this system show mutagenic activity. An even more recent tabulation of chemicals tested with the Salmonella assay system by Ames and his colleagues, using two new tester strains TA98 and TA100, shows that out of 175 chemical carcinogens tested 89% are mutagens (B.N. Ames, pers. commun.). Furthermore, tests on about 106 chemicals selected at random (including presumed non-carcinogenic structural analogs of known chemical carcinogens) show that only about 14% are mutagens (B.N. Ames, pers. commun.). Furthermore, when the chemicals structurally related to known carcinogens are eliminated, the percentage of chemicals which are mutagens is even lower (B.N. Ames, pers. commun.). As you might expect in such cases, the carcinogenic activity of the compounds giving a positive test for mutagenicity is not known. Obivously, these compounds need to be tested in longterm tests for carcinogenic activity in laboratory animals as well as in mutagenicity tests with other experimental organisms to determine the validity of this new approach. In this connection, mutagenic chemicals structurally related to known carcinogens should be tested for purity to ensure that the positive tests are not due to contamination of the sample being tested with a known mutagenic carcinogen. Implications for testing programs The basic question is then, whether these short-term tests which have been developed and validated in tests on several hundred chemicals, should be used on an even broader scale. With this approach, thousands of untested environmental chemicals could be assayed to try to identify those agents in our envir o n m e n t with potential mutagenic and carcinogenic affects on man. We are not ready, however, to extrapolate directly from data obtained in short-term tests for mutagenicity directly to man. This is simply because we do not have a sufficiently large data base to be confident that a genetically active c o m p o u n d in the short-term tests will produce significant levels of genetic damage in man. Because of the lack of such data 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, but also to determine the rate of increase over the spontaneous background for each class of genetic alteration. The general consensus is that these newly developed short-term tests provide a mechanism for alerting us to potential mutagenic and carcinogenic agents and that they are most effectively used at present to establish priorities for testing in higher organisms. There is concern that any battery of tests used as a prescreen may not be completely comprehensive and that some percentage of the chemicals tested will yield false negative and false positive results. Of particular concern are the false negatives, if no further testing is planned. To eliminate false negatives, a
173 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. To make the assay even more comprehensive perhaps a more thorough evaluation should be made of the use of repair-deficient strains of various microorganisms. A large number of such strains exist, but very few have been studied to determine their specificity and general utility as tester strains in mass screening programs. The addition of other strains as testers will be required to ensure that the short-term tests will detect any type of genetically active compound. We also need to know to what extent any battery of tests used as a prescreen will yield false positives. These are chemicals which will give positive results in the short-term test but not in higher organisms. 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 be able to determine the efficacy of the short-term tests and to validate this new approach. Future course The committee 17 Report of the Environmental Mutagen Society on Enviromental Mutagenic Hazards which appeared in the February 14 issue of Science this year recommended strongly that "screening should be initiated as rapidly and as extensively as possible." As a result of the development of the short-term tests, we now have a means for rapid, comprehensive and inexpensive screening. We have been given an unprecedented opportunity to revolutionize traditional methods of toxicological evaluation and to eliminate disease-causing agents from our environment. This approach should enable us to prevent an increase in the frequency of various diseases known to have a genetic basis, as well as to reduce the frequency of cancers resulting from environmental and occupational exposure. The shortterm tests are not perfect, but no test system ever developed provides 100% ascertainment. With time we can undoubtedly perfect this approach so that the activity of chemicals which give a false negative test with present techniques will also be detected. The general utilization of this new technology will enable us to evaluate numerous untested chemicals in our environment and to evaluate their mutagenic and carcinogenic potential for man. The data from exploratory studies with these newly developed short-term tests clearly "indicate that we have a problem of great concern. The challenge presented to geneticists by the recognition of this problem can only be met effectively by the development of new theoretical and applied research programs. We not only need assays with a higher level of ascertainment that will n o t miss mutagenically active agents, but we also need better ways to evaluate the risk of exposure to active agents for man. Finally, we need better test methods that can be used on man himself to determine the frequency and type of damage produced by exposure to an active agent. This is a situation that will require great ingenuity, creativity and imagination to resolve these difficult problems. Our success may not only have a marked influence on the quality of life, but because we are concerned about effects that are transmitted from one genera-
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tion to another and which are a c c u m u l a t e d with time, our success m a y well determine the future and very existence o f man on this planet. Who a m o n g us can afford to ignore such a challenge?
References 1 A m e s , B.N., W.F. D u r s t o n , E. Y a m a s a k i a n d F.D. Lee, C a r c i n o g e n s are m u t a g e n s : a s i m p l e test s y s t e m c o m b i n i n g liver h o m o g e n a t e s for a c t i v a t i o n a n d b a c t e r i a for d e t e c t i o n , Proc. Natl. Acad. Sci. ( U S ) . , 70 (1973) 2281--2285. 2 A m e s , B.N., H . O . K a m m e n a n d E. Y a m a s a k i , Hair d y e s are m u t a g e n i c : i d e n t i f i c a t i o n of a v a r i e t y of m u t a g e n i c i n g r e d i e n t s , Proc. Natl. A c a d . Sci. (US)., 72, ( 1 9 7 5 ) 2 4 2 3 - - 2 4 2 7 . 3 B a r t c h , H., C. Malaveille a n d R. M o n t e s a n o , H u m a n , rat a n d m o u s e l i v e r - m e d i a t e d m u t a g e n i c i t y o f v i n y l c h l o r i d e in S. l y p h i m u u i u m strains, Int. J. C a n c e r , 15, No. 3 ( 1 9 7 5 ) , 4 2 9 - - 4 3 7 . 4 B a r t c h , H. a n d R. M o n t e s a n o , M u t a g e n i c a n d c a r c i n o g e n i c e f f e c t s of v i n y l c h l o r i d e , M u t a t i o n Res., 32 (1975) 93--114. 5 Bridges, B.A., T h e m u t a g e n i c i t y of c a p t a n s a n d r e l a t e d fungicides, M u t a t i o n Res., 32 ( 1 9 7 5 ) 3 - - 3 4 . 6 B r o c k m a n , H . E . , F.J. de Serres a n d W.E. B a r n e t t , Analysis of a d - 3 m u t a t i o n s i n d u c e d by n i t r o u s acid in a h e t e r o k a r y o n of N e u r o s p o r a cr¢*.ssa, M u t a t i o n Res., 7 ( 1 9 6 9 ) 3 0 7 - - 3 1 4 . 7 Bronkes, P. a n d F.J. de Serres, R e p o r t of the w o r k s h o p o n the m u t a g e n i e i t y of c h e m i c a l c a r c i n o g e n s , D e c e m b e r 9 - - 1 1 , 1 9 7 4 , H o n o l u l u , U.S.A., M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 5 5 - - 1 6 0 . 8 Clive, D., M u t a g e n i c i t y of t h i o x a n t h e n e s ( H y c a n t h o n e , L u c a n t h o n e , a n d f o u r I n d a z o l e d e r i v a t i v e s ) at the T K l o c u s in c u l t u r e d m a m m a l i a n cells, M u t a t i o n Res., 26 ( 1 9 7 4 ) 3 0 7 - - 3 1 8 . 9 C r o w , J . F . , C h e m i c a l risk to f u t u r e g e n e r a t i o n s , Scientist a n d Citizen, 10 ( 1 9 6 8 ) 1 1 3 - - 1 1 7 . 10 De Serres, F.J. cd., L o n g - t e r m t o x i c i t y of a n t i s c h i s t o s o m a l drugs., J. T o x . E n v i r o n . H l t h , 1 ( 1 9 7 5 ) . 11 De Serres, F.J. a n d H.V. Malling, G e n e t i c analysis of a d - 3 m u t a n t s of N e u r o s p o r a crassa i n d u c e d b y e t h y l e n e d i b r o m i d e - - A c o m m o n l y used p e s t i c i d e , EMS N e w s l e t t e r , No. 4 ( 1 9 7 1 ) 3 6 - - 3 7 . 12 D r a k e , J.W., S. A b r a h a m s o n , J . F . C r o w , A. H o l l a e n d e r , S. L e d e r b e r g , M.S. L e g a t o r , J.V. Neel, M.W. S h a w , H.E. S u t t o n , R.C. y o n Borstel a n d S. Z i m m e r i n g , E n v i r o n m e n t a l m u t a g e n i c h a z a r d s , Science, 187 ( 1 9 7 5 ) 5 0 3 - - 5 1 4 . 13 D u c a t m a n , A., K. H i r s c h h o r n a n d l.J. Selikoff, V i n y l chloride a n d h u m a n c h r o m o s o m e a b e r r a t i o n s , M u t a t i o n Res., 31 ( 1 9 7 5 ) 1 6 3 - - 1 6 8 . 14 F u n e s - C r a v i o t o , F., B. L a m b e r t , J. L i n d s t e n , L. E h r e n b e r g , A . T . N a t a r a j a n a n d S. O s t e r m a n - G o l k a x s , C h r o m o s o m e a b e r r a t i o n s in w o r k e r s e x p o s e d to vinyl c h l o r i d e , L a n c e t , I, No. 7 9 0 4 ( 1 9 7 5 ) 4 5 9 . 15 Gentile, J.M. a n d M.J. Plewa, A bioassay for s c r e e n i n g h o s t - m e d i a t e d p r o x i m a l m u t a g e n s in agricult u r e , M u t a t i o n Res., 31 ( 1 9 7 5 ) 319. 16 Iida, S., M. l n o u e , K. Kai, N. K i t a m u r a , I. K u d o , M. S o n o , T. T s u r v o , H. H a y a t s o , A. Miura a n d Y. W a t a y a , S o m e p r o p e r t i e s of the d a m a g e of D N A a n d p h a g e l a m b d a i n d u c e d b y bisulfite, M u t a t i o n Res., 26 ( 1 9 7 4 ) 4 3 3 - - 4 3 4 . 17 I k e d a , Y., S. H o r i u c h i , T. F u r u y a , O. U c h i d a , K. Suzuki a n d J. A z e g a m i , I n t e r i m R e p o r t : I n d u c t i o n o f Gastric T u m o r s in Mice by F e e d i n g o f F u r y l f u r a m i d e , F o o d S a n i t a t i o n S t u d y Council, Minister of H e a l t h a n d Welfare, J a p a n , A u g u s t ( 1 9 7 4 ) . 18 K a d a , T., E s c h e r i c h i a coli m u t a g e n i c i t y of f u r y l f o r a m i d e , J a p a n . J, G e n e t . , 48 ( 1 9 7 3 ) 3 0 1 - - 3 0 5 . 19 Kiese, M. a n d E. R a u s c h e r , T h e a b s o r p t i o n of p - t o l u e n e d i a m i n e t h r o u g h h u m a n skin in hair d y e i n g , T o x i c . A p p l . P h a r m a c . , 13 ( 1 9 6 8 ) 3 2 5 - - 3 3 1 . 20 K o n d o , S. a n d H. I c h i k a w a - R y o , T e s t i n g a n d classification o f m u t a g e n i c i t y of f u r y l f u r a m i d e in E s c h c r i c h i o coli, J a p a n . J. G e n e t . , 48 ( 1 9 7 3 ) 2 9 5 - - 3 0 0 . 21 L o p r i e n o , N., R. Barale, S. Baroncelli, C. Bauer, G. B r o n z e t t i , A. C a m m e l l i n i , G. Cercignani, C. Corsi, G. Gervasi, C. L e p o r i n i , R. Nieri, M. Rossi, G. S t r e t t i a n d G. T u r e h i , E v a l u a t i o n of the g e n e t i c e f f e c t s of v i n y l c h l o r i d e m o n o m e r (VCM) u n d e r the i n f l u e n c e of liver m i c r o s o m e s (in press). 22 Mailing, H . V . , D i m e t h y l n i t r o s a m i n e : f o r m a t i o n of m u t a g e n i c c o m p o u n d s b y i n t e r a c t i o n w i t h m o u s e liver m i c r o s o m c s , M u t a t i o n Res., 13 ( 1 9 7 1 ) 4 2 5 - - 4 2 9 . 23 Marshall, S. a n d W.S. P a l m e r , D a r k u r i n e a f t e r hair c o l o r i n g , J. A m e r . Med. Ass., 2 2 6 ( 1 9 7 3 ) 1 0 1 0 . 24 McCalla, D.R. a n d D. V o u t s i n o s , On the m u t a g e n i c i t y of n i t r o f u r a n s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 3 - - 1 6 . 25 M c C a n n , J. a n d B.N. A m e s , A s i m p l e r m e t h o d for d e t e c t i n g e n v i r o n m e n t a l c a r c i n o g e n s as m u t a g e n s , A n n . N.Y. A c a d . Sci., 2 6 9 ( 1 9 7 5 ) 2 1 - - 2 5 . 26 M c C a n n , J., N.E, S p i n g a r n , J. K o b o r i a n d B.N. A m e s , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s : b a c t e r i a l t e s t e r strains w i t h R f a c t o r plasmids, Proc. Natl. A e a d . Sei. ( U S ) , 72 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 . 27 Ong, T. a n d F.J. de Serres, M u t a g e n i c e v a l u a t i o n of a n t i s c h i s t o s o m a l a g e n t s in N e u r o s p o r a crassa, J. T o x . E n v i r o n m . H l t h , 1 ( 1 9 7 5 ) 291, 28 Ong, T.M. a n d S h a h i n , M.M., M u t a g e n i c a n d r e c o m b i n o g e n i c activities o f t h e f o o d a d d i t i v e f u r y l f u r a m i d e in e u k a r y o t e s , Science, 1 8 4 ( 1 9 7 4 ) 1 0 8 6 - - 1 0 8 7 .
175 2 9 P u r c h a s e , I . F . H . , C . R . R i c h a r d s o n , D. A n d e r s o n , C h r o m o s o m a l a n d d o m i n a n t l e t h a l e f f e c t s o f v i n y l c h l o r i d e , T h e L a n c e t , II, N o . 7 9 3 1 ( 1 9 7 5 ) 4 1 0 - - 4 1 1 . 3 0 R a n n u g , V., A. J o h a n s s o n , C. R a m e l a n d C . A . W a c h t m e i s t e r , Tile m u t a g e n i c i t y o f v i n y l c h l o r i d e a f t e r metabolic activation, Ambio, 3 (1974) 194--197. 31 R a p o p o r t , I . A . , N . N . Z o z , S.I. M a k a r o v a a n d T . V . S a l n i k o v a , S u p e r m u t a g e n s ( N a u k a , M o s c o w , 1 9 6 6 ) . 3 2 Searle, C.E., D . G . H a r n d e n , S. V e n i t t a n d O . H . B . G y d e , C a r c i n o g e n i c i t y a n d m u t a g e n i c i t y t e s t s o f some hair colourants and constituents, Nature, 255 (1975) 506--507. 3 3 S l a t e r , E . E . , M.D. A n d e r s o n a n d H.S. R o s e n k r a n z , R a p i d d e t e c t i o n of m u t a g e n s a n d c a r c i n o g e n s , C a n c e r R e s . , 31 ( 1 9 7 1 ) 9 7 0 - - 9 7 3 . 3 4 T a z i m a , Y., T. K a d a a n d A . M u r a k a m i , M u t a g e n i c i t y o f n i t r o f u r a n d e r i v a t i v e s , i n c l u d i n g f u r y l furamide, a food preservative, Mutation Res., 32 (1975) 55--80. 3 5 T o n o m u r a , A . a n d M.S. Sasaki, C h r o m o s o m e a b e r r a t i o n s a n d D N A r e p a i r s y n t h e s i s in c u l t u r e d h u m a n cells e x p o s e d t o n i t r o f u r a n s , J a p a n . J. G e n e t . , 4 8 ( 1 9 7 3 ) 2 9 1 - - 2 9 4 . 3 6 U c h i d a , O., T. F u r u y a , K. K a w a m a t a , S. H o r i c u c h i , Y. I k e d a , K . S u z u k i a n d J. AT.egami, S t u d i e s o n t h e t o x i c i t y o f f u r y l f u r a m i d e ( A F - 2 ) . II. S t u d y o n the c a r c i n o g e n i c i t y in m i c e , J a p a n . J. P h a r m a c . (in press). 3 7 V a l e n c i a , R., S. A b r a h a m s o n , P. W a g o n e r a n d L. M a n s f i e l d , T e s t i n g f o r f o o d a d d i t i v e - i n d u c e d m u t a t i o n s in Drosophila melano¢aster, M u t a t i o n R e s . , 21 ( 1 9 7 3 ) 2 4 0 - - 2 4 1 . 3 8 W e e k e s , V., J. Williams, P. B u t l e r , G . R o y a n d D. B r u s i c k , R a p i d s c r e e n i n g t e c h n i q u e s f o r m u t a g e n icity e v a l u a t i o n u s i n g m i c r o b i a l cells c o m b i n e d w i t h m a m m a l i a n a c t i v a t i o n f r a c t i o n s , M u t a t i o n Res. (in press). 3 9 Y a h a g i , T., M. N a g a o , K. H a r a , T. M a t s u s h i m a , T. S u g i m u r a a n d G . T . B r y a n , R e l a t i o n s h i p s b e t w e e n t h e c a r c i n o g e n i c a n d m u t a g e n i c o r D N A - m o d i f y i n g e f f e c t s o f n i t r o f u r a n d e r i v a t i v e s i n c l u d i n g 2-(2furyl)-3-(5-nitro-2-furyl) acrylamide, a food additive, Cancer Res., 34 (1974) 2266--2273.
