3futation Research, 29 (1975) 295-3 °0 C~ Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands




Genetics Toxicology Branch, Division of Toxicology, Food and Drug ddministratio~2, Department of Health, Education and Welfare, Washington, D.C. 2o2o4 (U.S.A.) (Received November 4th, I974)


A brief account is given of the history of the legislative acts that give responsibility to the U.S. Food and Drug Administration (FDA) for ensuring the safety of foods, drugs, and cosmetics. Within the present legislative framework the FDA has the authority to impose regulations which are designed to ensure the safety of all foods, drugs, and cosmetics. The existing legislative authority is adequate for this purpose; however, the difficulty lies instead with technology and the inadequacy of scientific perspective in the emerging area of mutagenicity testing. Earlier efforts in development of mutagenicity screening systems culminated only a few years ago in the proposal to use the host-mediated assay, somatic cell cytogenetics, and dominant lethal tests collectively. Subsequent research efforts indicated that there were serious practical and scientific deficiencies in using this approach. More recently a new proposal, the tier system, has been suggested as an alternative measure. The proposed tier system at FDA consists of three testing levels of increasing complexity. The first tier is an initial screening effort using techniques having maximum sensitivity that are also useful fo~ large-scale, rapid testing. The second tier is designed to identify and confirm that the presumptive mutagens detected in the first tier are truly mutagenic for higher organisms, most especially, for mammals. The third tier would be devoted to explicit genetic tests in mammals designed to ascertain the imposed risk to man by the introduction of a mutagen in our environment. The FDA is currently involved in a number of research activities in the area of mutagenicity safety screening which will explore the adequacies and possible deficiencies of the tier system approach. These efforts are described for our in-house activities, our contract activities, and our cooperative and collaborative activities with other government agencies and institutions. Over the years, the Congress of the United States has passed legislation intended to protect the public from unwholesome and adulterated foods; to ensure the safety of food and cosmetic products; and to ensure the efficacy and safety of drugs. The



M A Y E R , W. G. I:I_\3IM

first of these bills was passed by Congress and becaine law in I0O0, nearh" 7° year> ago, and led eventually to tile establishment of the Food and Drug Administration which is now responsible for the implementation of these law>. During the c~mr~e ,,f more than 3 decades of implementation, certain faults and shortconlings of the I0o~ law were bcought to light and in 1038 a second law was enacted which considerabh strengthened governmental control over food% drugs, and c~smetic,. In i05 S, tlle Food Additives Amendment was added to the Federal Food, Drug, and ('~smetic Act. This amendment placed the primary responsibility for assuring the safet\" of all food additives on the producers and uqers of food additives. The previ~,us I938 law only provided requirements for pre-marketing clearance ~f drugs for safet\. However, as a result of the 1958 Amendment, toxicologic testing prior to marketing becamc required of all regulated food additives to ensure their safety for their intended u>e. Additionally, detailed accounts are required t~J be presented to the [:DA in the form of petitions and must include such information as the chemical identity of the substance, hmv it is m'mufactured, and adequate analytical meth~,ds for its detection and measuremeilt iu the food supply at the levels of proposed use. The petitioner must further establish that the additive will accomplish the intended phy.,ical ,~r technical effect and that the proposed level for use is no higher than that necessary to accomplish the desired effect. This law was followed by similar preclearance requirement., for colors. In 1062 the drug laws of 1038 were strengthened bv requiring that drugs be effective as well as safe. Cosmetics at this time require no preclearance for safety ,~r efficacy. In addition to these historic legislative acts, the Congress -f the United State> is presently considering proposed legi_:lation known as the Toxic Substances Control Act. The intention of this legislation is to emp~wer the Environmental Protecti(m Agency to regulate chemical substances to which man and his environment are exposed b\" their manufacture, distribution, u-e, or disposal and which may po.~e an unreasonable risk to human health and the environment. Enactment of this propo.-ed legislation would give authority to the Environnlental Protection Agency to write regulations to establish standards for test protocols which will >erve to provide meaningful and relevant data for the protection of human health and the environment. Regulations written for the purpose of implementing this legislation w~mld apply- to those substances which are produced in commercial quantities but would exclude certain materials that are regulated by other existing acts ,~f Omgress. l:~)r example, foods, drugs, and cosmetics are subject t~, regulation by the Federal Food, Drug, and Cosmetic Act and would be exempt from the propo>ed T.xic Substances Control Act. The current and proposed legi.,lation mentioned here suffice to empower appropriate governmental agencies with the authority to pronmlgate regulations designed to ensure safety and health by using the most relevant and effective technology available, including mutagenicity te>ting. Thu>, the encumbrance to promulgating requirelnents applicable to certain emerging areas of toxicology..~uch as mutagenesis, is not one of legislative authority; the difficult3", instead, lies with technology and the adequacy of .~cientific knowledge and interpretation. The difficulty concerns the current needs for practical appr-aches which effectively address the regulatory i>>ues and provide answers which serve to protect public health. In the area of mutagene.,is, as in any other area of toxicology, a sufficient data base must be built



and sufficient experience must be acquired with test systems before an assessment can be made of their practicability, predictive value, and biologic validity. The FDA, the Environmental Protection Agency, and the Atomic Energy Commission in cooperation with the National Institutes of Health have instituted and funded numerous programs to expedite the development of knowledge and facility necessary for writing guidelines which call for mutagenicity testing. These programs include basic and applied research, collaborative studies with industry, academia and government, data storage and retrieval systems, monographs, workshops, conferences, and symposia. It is evident that such tests must meet certain criteria if they are to be considered for implementation within a regulatory context. Such tests must be reproducible. They must be practical in the context in which the test is proposed for use. That is, the time required and the level of effort required for conduct of the test, as well as the availability of laboratories possessing the necessary skill and expertise for testing, must be considered. Such tests must further measure an effect which is either closely or directly related to genetic events which are of concern to man. Ideally we would want tests that are highly sensitive, tests that always register positive for substances that might pose a genetic damage threat to man, tests that are simple and inexpensive to conduct, and finally tests that will provide an assessment of risk or safety to the exposed population. It has been widely held for a long time by advocates of mutagenicity testing that there is no single test which can satisfy all of these criteria and it is widely realized, therefore, that a battery of tests must be employed if we are to effectively assess chemical mutagenic potential. Earlier efforts in the development of mutagenicity screening systems culminated only a few years ago in the proposal to use three systems simaltaneouslyX,l°,12,14; namely, somatic cell cytogenetics 15, dominant lethality 1, and the host-mediated assay 13. It was thought at the time that no mutagen would escape detection in at least one of the three systems. Problems with this approach, however, became apparent through testing efforts within and for the FDA as well as in other laboratories. For example, agents which function through intercalation of DNA and require metabolic activation apparently register negative in all three test systems. Such is the case with the potent carcinogen, 2-acetylaminofluorene4, 2~. Other aromatic amines requiring metabolic activation for their genetic activity fail to produce a positive response in the host-mediated assay 2'. It remains to be seen whether future levels of experimental sophistication can permit such compounds to register positive in these systems. In a number of cases the host-mediated assay has been found to be a relatively insensitive procedure for chemical mutagenesis monitoringT, s, and some of the factors influencing the response have been explored by ZEIGER22. Some potent mutagens which do not require metabolic intervention for their activity required inordinately high concentrations to produce a minimal positive effect in the host-mediated assay compared with similar in vitro tests3, ~. Hycanthone, a direct-acting mutagen effective in vitro, is in fact negative in the host-mediated assay 3 presumably because of the insensitivity of the method. In other instances, the dominant lethal test has registered negative for some recognized mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine ~, 5-bromodeoxyuridine 4, dimethylnitrosamine4,1% and nitrogen mustard ~. Conficting data exist as to the dominant lethality of other substances such as caffeine*,n, hycanthoneg, 20, DDT4, TM, and triflupromazine~7, TM. Finally, a number of substances, which are alkyl-



MAYER, W. (,. I:t.AM3I

a t i n g agents able to i n t e r a c t with D N A ~ a n d therefore might he suspected ~Jf having m u t a g e n i c potential, are negative in the d o m i n a n t lethal test. Such is the case fc~r d i m e t h v l sulfate, epichlorohydrin, e t h y l e n e ehlorohydrin, a n d t r i e t h v l p h o s p h a t e L W e therefore face the d i l e m m a of w h e t h e r such results are the reflection of test .-v~tem i n a d e q u a c y or w h e t h e r m a m m a l i a n organism~ are c o n s i d e r a b l y less sensitive t l n u t a t i o n a l insult t h a n had been feared. W e cannot, at present, decide hetween the.,e two a l t e r n a t i v e s because we have r e l a t i v e l y little information to d r a w upon t . m a k e a decision. We will not know for some t i m e the precise relevance of either n o n - m a m malian tests or tests in m a m n l a l s which measure end points other t h a n m u t a t i o n a m o n g p r o g e n y of t r e a t e d animals. In the m e a n t i m e it will be nece.~sarv t~ proceed b y using, as in the past, all of the basic genetic a n d molecular genetic i n f o r m a t i o n a v a i l a b l e to devise b e t t e r a n d lnore e x p e d i e n t ways of identifying p o t e n t i a l genetic hazards. It is also becoming increasingly clear t h a t the p r o b l e m of te~ting h u n d r e d s or p e r h a p s t h o u s a n d s of substances t h a t are a p a r t of inan's e n v i r o n m e n t is not nlanageable t h r o u g h the a p p l i c a t i o n of tile three p r e v i o u s l y m e n t i o n e d test systelns. Thus it is a p p a r e n t t h a t a m e t h o d m u s t be devised for the setting of priorities in m u t a g e n icity testing. This becomes especially relevant if we desire a n d accept t h a t m a m m a l i a n test systems, which are i n h e r e n t l y more involved, expensive, a n d time consuming, will p l a y a inajor role in n m t a g e n i c i t y testing. Such tests can onh" effectiveh" be e m p l o y e d if t h e y are used judiciously a n d selectively. W i t h i n the pa.~t y e a r a new a p p r o a c h to the setting of prioritie~ for m u t a g e n i c i t y testing has been suggested, first b y BRIDt;ES ~ of E n g l a n d at a workshop on the evaluation of cheinical n m t a g e n i c i t y d a t a in relation to p o p u l a t i o n risk held at the Research Triangle Park, N o r t h ( ' a r o l i n a a n d second b y FLA-~tM6 of the VDA at the First I n t e r n a t i o n a l Conference on E n v i r o n m e n t a l Mutagens held at Pacific Grove, California. This a p p r o a c h is known as the tier s y s t e m a n d consists of three levels of increasing c o m p l e x i t y , with tile testing at each tier designed for different purposes a n d t~) answer different questions. The sinlplest a n d first level of this plan would b : an initial screening effort in which a n y a n d all p o t e n t i a l m u t a g e n s would be identified. Ideally, there should be limited allowanc:, for false negatives while the occurrence of fal.,e positives could be t o l e r a t e d but would hopefully be mitfimal. Such t e 4 s mu~t in a d d i t i o n be rapid, inexpensive, easy to perform, a n d r e a d i h ' reproducible. Clearly, o n h te~ts in lnicroorganisins can fulfill the.~e requirements. We inu.-t be further concerned, however, t h a t we can d e t e c t in inicrobes all the genetic events of iinportance to man a n d als~ t h a t m a m l n a l i a n m e t a b o l i s m is sufficiently p r o v i d e d a n d a c c o u n t e d for in these test~. The second tier or second level of effort would serve to identify a n d c~nfirm t h a t the p r e s u m p t i v e m u t a g e n , d e t e c t e d ill tier I were t r u l y m u t a g e n i c for higher organisms and, most especially, for m a m m a l s . It is a s s u m e d t h a t testing m e t h o d s in tier 2 can be more involved a n d complex, since we a n t i c i p a t e t h a t there will be r e l a t i v e l y fewer compounda which need testing, a~ c o m p a r e d with tier I. Here, tebt_, ill m a m m a l i a n cell culture or in D r o , o p h i l a would be a p p r o p r i a t e . Drosophila shares m a n y of the same metabolic .,vstem~ with m a m m a l s a n d it is t h o u g h t t h a t cell culture~ could be s u p p l e l n e n t e d ill some fashion with liver microsome fractiona, ~)r the like, .,ilnilar t~, the microhial bvsteln~ in tier t. The second tier might a g o include testing f(~I heritable t r a n s l o c a t i o n s in mice. It would be especially apprc,priate at the tier 2 level t~) have tests involving meiotic as well a~ mitotic cells.



The third tier would be devoted to explicit genetic tests in mammals for the purpose of ascertaining the imposed risk to man by the presence of a particular mutagen in some sector of our environment. It is anticipated that only some of the compounds that were positive in tier 2 will require testing in tier 3. Tier 3 testing therefore would be designed to test those compounds which are either unavoidable or are considered sufficiently important that they must be used. In such cases we need some realistic estimate of the associated genetic risk. At present mammalian tests which would be useful in tier 3 are considerable undertakings. The specific locus test in mice developed by m a n y workers as well as the heritable translocation test are being considered for use in tier 3. It is additionally necessary that new mammalian tests are developed which will measure gene mutation but without the enormous effort now required. There is evidence that such tests are indeed forthcoming but realistically they are yet several years in the future. The FDA has has already begun active consideration of the tier system as a viable means of effectively screening the mutagenic potential of chemicals. In our ongoing efforts to evaluate food additive compounds for their genetic activity, we have begun testing some substances at the tier z and tier 2 level by using in the latter instance Drosophila melanogaster, heritable translocation in mice and mammalian cells in tissue culture. These activities are being carried out within the laboratories of the FDA and are also being funded by contract with the FDA. Tier I efforts to be carried out over the next year include the testing of approximately 60 compounds selected from the GRAS (generally recognized as safe) list. These compounds will be tested in Salmonella typhimurium reverse mutation strains developed by BRUCE AMES. They will be further tested in diploid Saccharomyces eerevisiae strains developed by FRIEDRICH ZIMMERMANNfor the detection of induced mitotic recombination. In addition to in vitro direct treatment tests, mammalian metabolism will be provided in other tests by the use of cell-free homogenates and microsomal preparations of liver, lung and testes from three animal species. Our efforts with Drosophila melanogaster for tier 2 are only beginning and therefore somewhat more limited in scope. In tile forthcoming year, four compounds will be tested for their ability to induce sex-linked recessive lethals and chromosome breakage and rejoining in the fruit fly. Exploratory efforts using mammalian cell cultures are being conducted to search for additional markers which can be used as genetic indicators of chemical mutagenesis. Work with the heritable translocation test in mice has begun both in the FDA laboratories and in contract laboratories. Our efforts for the present are confined to gaining basic experience with the test system itself. Other efforts are designed to compare directly the utility of the translocation test with that of the dominant lethal test. Further studies are designed to determine whether the indirect test, that is, the measurement of reduced litter size in the F1 generation is a valid criterion for the assessment of induced heritable translocations. Success in these areas is then expected to permit us to begin choosing compounds, found to be positive in our tier I screening effort, to be tested for their ability to induce heritable translocations within our tier 2 testing program. The National Cancer Institute, using the contract mechanism, is presently engaged in the testing of 85 compounds, most of them known carcinogens, for their capacity to induce genetic damage. The purpose of this effort is to determine the extent to which mutagenicity screening techniques are of predictive value in the




of carcinogenic


the host-mediated



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are being used

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point mutation


Scientific personnel

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REFERENCES I BATEMAN, A. J., AND S. S. EPSTEIN, D o m i n a n t lethal m u t a t i o n s in nlamnlal.,, in .\. HOLLAENDER (Ed.), Chemical Mutagens: Pmnciples and 3Iethods /:or their Detection, Vol. 2, Plentnn, New York, I97 t, pp. 541-568. 2 BRIDGES, B. A., S o m e general principles of n l u t a g e n i c i t y screening a n d a possit)le l r a m e w o r k for t e s t i n g procedures, Environ. Health Persp., 6 11073 ) 22t-227. 3 BRUSICK, D. J., AND \'. W. MAYER, New d e v e l o p n l e n t s in m u t a g e n i c l t y ~creenmg t e c h n i q u e s w i t h yeast, Environ. Health Persp., 6 (t973) 83-06. 4 EPSTEIN, S. S., E. ARNOLD, J. ANDREA, \\r. BASS AND Y. BISHOP, Detection of chemical rot> t a g e n s b y t h e d o m i n a n t lethal a s s a y ill t h e mouse, Toxicol. Appl. Pharmacol., 23 (I972) 2,~;,";325 • 5 FISHBEIN, L., \V. (;. FLAMM AND H. l.. I?ALK, Chemical 31tttagens: Envlro~zmc~ttal t~fi)'cts ,,~ Biological Systems, Academic Press, New York, 107o. 6 FLAMM, \V. G., A tier sy'stem a p p r o a c h to m u t a g e n testing, 3lutation Res., 26 (19741 329 333. 7 FROHBERG, H., AND A. BAUER, M n t a g e n i c i t y trials u n d e r toxicological aspects, Arcneim. Forsch., 23 (I973) 23o-236. S FROIfBERG, H., AND ~[. SCHULZE-SCHENCKING, Recent findings c o n c e r n i n g (l,~c respollse r e l a t i o n s h i p in m u t a g e n i c i t y t e s t i n g of chemical~, Arch. Toxicol., 32 {I974) r - t 7 . 9 GREEN, S., J. v . CARR, F. M. SAURO AND M. S. LEGATOR, Effects of h y c a n t h o n e in spernlatogonial cells, D N A s y n t h e s i s in bone m a r r o w a n d d m n i n a n t l e t h a l i t y in rats, ./. Pharmacol. Exp. Ther., I87 (I073) 437-443IO HOLLAENDER, A. (Ed.), Conclusion, in Chemical 3lutagens: Principles and Methods lor lh,.tr Detection, Vol. 2, P l e n u m , New York, i97 I, pp. 607 61o. I I I(UHLMAN, \V., H. I?ROMME, E. HEEGE AND \V. OSTERTAG, T h e m u t a g e n i c action of caffeine in h i g h e r o r g a n i s m s , Cancer Res., 28 (I968) 2375 "389. I2 LEGATOR, 3,[. S., Deficiencies in o u r p r e s e n t protocol for chemical e v a l u a t i o n a n d possible remedies, A n n . N . Y . Acad. Sci., 179 11071) 5o8 513 . 13 LEGATOR, M. S., AND H. V. MALLING, T h e h o s t m e d i a t e d assay, a practical p r o c e d u r e for e v a l u a t i n g p o t e n t i a l m u t a g e n i c a g e n t s in n m m m a l s , in A. HOLLAE.NDER (Ed.I, Chemical 3lutagens: Principles a~d Methods lbr their Detectzoll, Vol. 2, P l e n u m , New York, I97 I, pp. 569-580. r 4 I\IRAK, E..'~[., (Chairman}, Report o_ltht Secretary's Commission on Pesticuha and Thcw Rdati,mship to Environmental Health, U.S. D e p a r t m e n t of Health, E d u c a t i o n a n d \Velfare, \ V a s h i n g t o n , D.C., I969. 15 NICHOLS, \V. \V., P. ~IOORHEAI) AND G. BREXVER, C h r o n l o s o m e m e t h o d o l o g i e s in m u t a g e n testing, Ad Hoc c o n l m i t t e e report, To.ricol. Appl. Pharmacol., 22 11972) 269-275. I 6 PALMER, K. A., S. GREEN AND ~l. S. LEGATOR, D o n i i n a n t lethal s t u d y of p , p ' - l ) l ) T in rat>, Food Cosmet. To.ricol., 1 t 11973) 53-62. 17 PETERSEN, K. \V., .aND 3i. S. LEGATOR, D o m i n a n t lethal effects of trifluproinazine, Mutatio, Res., 17 (Ic~73) 87-92. iS RaY, V., H. E. HOLDEN, J. H. ELLIS AXD M. L. HYXECK, A s t u d y of triflupronlazine in domin a n t - l e t h a l , c y t o g e n e t i c a n d h o s t m e d i a t e d a s s a y s , 3Iutataon Res., 18 (1973) 3 ° t-3o9. 10 SAURO, F. M., L. FRIEDMAN AND S. GREEN, Biochemical, m u t a g e n i c a n d pathological effects of n i t r o s a n l i n e s in rats, Toxlcol. Appl. Pharmacol., 23 (I973) 4492o "WHO COMMITTEE, Report on Hycanthone, \VHO, G e n e v a , t97 I. 2t ZEIGER, E., P e r s o n a l c o m m u n i c a t i o n . 22 ZEIGER, E., Some factors affecting t h e h o s t - n l e d i a t e d a s s a y response, Era,iron. Health Persp. 6 {'I973) t o I - I O 0.

Legislative and technical aspects of mutagenicity testing.

A brief account is given of the history of the legislative acts that give responsibility to the U.S. Food and Drug Administration (FDA) for ensuring t...
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