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Mutation Research, 38 (1976) 177--190 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
PREDICTIVE VALUE OF MUTAGENICITY TESTS IN CHEMICAL CARCINOGENESIS
H. B A R T S C H International Agency for Research on Cancer, Unit o f Chemical Carcinogenesis, 150 cours Albert Thomas, 69008 Lyon, France
{Received October 10th, 1975) {Accepted November 28th, (1975)
An essential role of environmental factors in the aetiology of human cancer has been shown by the unequivocal evidence of the chemical origin of occupational cancer, as in the cases of urinary bladder tumours in workers exposed to aromatic amines [29,13,12] lung cancers in workers exposed to bis(chloromethyl) ether [30] or angiosarcomas of the liver in workers exposed to vinyl chloride [31] ; the well
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F i g . 2. L i v e r - e n z y m e - m e d i a t e d m u t a g e n i e i t y ( h i s + r e v e r t a n t c o l o n i e s o f S. t y p h i m u r i u m 1530/plate) of BD-VI rat tissue and individual human biopsy samples, represented by different characters. N-nitrosamine w e r e a s s a y e d a c c o r d i n g t o A m e s e t al. [ 3 ] , a n d v i n y l c h l o r i d e a s d e s c r i b e d b y B a r t s c h e t al. [ 7 ] . T h e c o n c e n t r a t i o n o f t h e c a r c i n o g e n s u s e d w a s w i t h i n a l i n e a r r a n g e o f t h e d o s e - r e s p o n s e c u r v e s o b t a i n e d i n a n assay system containin~ 9 000 ~ supernatant equivalent to 39 mg liver/plate. Number of spontaneous his + revertant colonies from appropriate controls (no co-factors or no substrate) was subtracted. Data from B a r t s c h e t al. [ 9 ] a n d u n p u b l i s h e d w o r k .
metabolites, but, as measured for individual Z, several carcinogens, which even belong to different chemical classes. This has been shown for five different compounds (Fig. 2). The identification of people who may be at exceptionally high risk for cancer because of a genetically determined enzyme profile may in future be of some help in establishing regulatory measures to limit the levels of carcinogens in the environment. Such levels should be established by taking into consideration the possible risk for the most susceptible individuals. The last part of this discussion concerns the usefulness of these mutagenicity tests in predicting the possible carcinogenic effects of chemicals in man. Despite the converging tendency of chemicals to be both carcinogenic and mutagenic, it is not known at present whether in future all carcinogens will be found to be mutagens and all mutagens carcinogenic. For the strong mutagens, such as base analogues, nitrous acid and hydroxylamine, no carcinogenic effect in animals has been reported so far [48]. These chemicals do not act via electrophilic intermediates. On the other hand, synthetic steroidal sex hormones, which are carcinogenic in animals [31], have not yet been reported to be mutagenic, and we have also been unable in our own laboratory to detect a mutagenic effect in tissue-mediated assays in vitro using the human and animal carcinogen a,a'-di-
185
ethylstilboestrol as substrate with the T A 1 0 0 strain of S. typhimurium (Bartsch, unpublished). These findings would suggest that for this class of compounds, a different cancer-inducing mechanism may be implied. There are various limitations of the mutagenicity test systems, since some of the factors that determine the processes of cancer development in vivo cannot be duplicated in, for example, mutagenicity systems in vitro. Some of the most important of these determining factors are the concentration of ultimate reactive metabolites available for reaction in organs and animal species with cellular macromolecules, which is a consequence of a critical balance between metabolic activation and detoxication processes; an organ-specific release of proximate or ultimate carcinogens by enzymic deconjugation; the biological half-life of metabolites; and organ-specific D N A repair or frequency of D N A replication in target or non-target organs and immuno-surveillance. The use of mutagenicity tests in the assessment of the carcinogenic risk due to chemicals justifies their availability, since, although an enormous number of new chemicals is entering the environment, the world capacity for testing for carcinogenicity is only about 500 compounds per year, and it is not possible to test all of them in long-term carcinogenicity tests [ 3 2 , 3 3 , 3 4 , 3 5 , 3 6 ] . It is necessary, therefore, to pre-screen in order to establish priorities for long-term testing. In evaluating these tests, four points should be emphasized: (1) positive results from mutagenicity tests given at level C {Table IV) cannot automatically be taken to imply a definite carcinogenic effect in man {level A); (2) positive or negative results from these tests cannot substitute for long-term carcinogenicity
TABLE
IV
FRAMEWORK
OF CARCINOGENICITY
Valid data o n
TEST PROCEDURES
Test s y s t e m
N o data o n
Carcinogenic in m a n
T h r e s h o l d dose; individual
T_ Target organ in man; high risk groups
Species and organ specificity d o s e response in animals
Mechanism o f m e t a b o l i c activation in animals and man; t y p e o f genetic damage
;
risk
A
Epidemiologicalstudies
l
Level
Positive
]
Predictive value for extrap o l a t i o n (at present limited); target organ; threshold d o s e
J
Careinogenieity test in animals I Positive
Mutagenicity tests Microbial, m a m m a l i a n , h u m a n cells/activation in vivo and in vitro Chemicals
Level
~ --
B
Species a n d / o r organ specificity correlation b e t w e e n mutagenic and carcinogenic p o t e n c y Level
C
;
186 testing in animals (level B); (3) the present methodology, in particular tissuemediated mutagenicity assays, is effective in predicting the carcinogenic potential of chemicals with a high probability, ranging at present between 80 and 90%, but gives no indication of target organs or species specificity of the carcinogenic action of the chemical; and, (4) the relative potency of a chemical as a mutagen cannot at present be correlated with its p o t e n c y as a carcinogen in experimental animals or man. Taking into account present reproducibility, cost, number of chemicals that can be examined in a short time and the scientific basis of the test, tissue-mediated mutagenicity procedures using well-characterized genetic indicators and a metabolically defined activation system in vitro (apart from the Drosophila test [63] ) appear at present to be the most promising short-term tests for the detection of adverse biological effects produced by chemicals. The present methodology still incurs the chance of false negative results, depending on the system used, either due to mutagen specificity, lack of appropriate cofactors for activation or to extreme reactivity and volatility of the c o m p o u n d or its metabolites [9]. However, the number of false negative results obtained in the tissue-mediated assay in vitro is very small in comparison with that obtained with other mutagenicity test procedures. As has often been mentioned, theoretical limitations are implied in the short-term tests currently used, because the development of cancer in vivo is determined by factors that cannot yet be duplicated in many of the mutagenicity test systems. Although there are still many problems involved in the interpretation of results of mutagenicity tests in terms of evaluating the carcinogenicity of chemicals, short-term tests can already be used in detecting possible cancer-causing agents with a sensitivity which did n o t exist ten years ago. They could thus be a powerful tool, when used in combination with epidemiological studies, in environmental control. This possibility, therefore, raises two important issues: (1) the validity of the short-term tests must be based on reliable corroboration by data from long-term tests in experimental animals; so, first of all the long-term data must be critically analysed and accepted to provide solid and definite background information; and, (2) the scientific community, as well as health authorities, must reach a concensus on the usefulness of results from these test systems in predicting possible carcinogenic effects of c o m p o u n d s in man. Otherwise, and if no public health measures are taken, for instance, to remove the agent from the environment or greatly to reduce exposure to it, cancer in man can only be treated and not prevented.
Acknowledgements
The author's experimental work was partially supported by the National Cancer Institute of the USA, Contract No. 1CP-55630. For valuable discussion and/or experimental assistance, the author is indebted to L. Tomatis, R. Montesano, C. Malaveille, A. Barbin, A. Camus, G. Planche, G. Brun and H. Br~sil. The help of Miss P. Stafford Smith and Mrs E. Ward in preparing the manuscript is gratefully acknowledged.
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Note added in proof Loprieno et al. [68] have observed a pronounced mutagenic effect of vinyl chloride in Schizosaccharomyces pombe and Saccharomyces cerevisiae, only when mouse-liver microsomes were added to the assays in vitro. Vinyl chloride was also active in the host-mediated assay in mice. References 1 A m e s , B.N. a n d J. M c C a n n , 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 t e s t s y s t e m , in R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s (eds.) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , L y o n ( I A R C S c i e n t i f i c P u b lications No. 12), 1976. 2 A m e s , B.N., A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s in u r i n e : s t u d i e s w i t h t h e c a r c i n o g e n 2a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. (Wash.), 71 ( 1 9 7 4 ) 7 3 7 - - 7 4 1 . 3 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d D . F . 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Miller, T h e m u t a g e n i c i t Y o f c h e m i c a l c a r c i n o g e n s : c o r r e l a t i o n s , p r o b l e m s a n d i n t e r p r e t a t i o n s , in A. H o l l a e n d e r ( e d . ) C h e m i c a l M u t a g e n s , Vol. 1, P l e n u m , N e w Y o r k , 1 9 7 1 , p p . 8 3 - 119. 4 9 Miller, J . A . , C a r c i n o g e n e s i s b y C h e m i c a l s : a n o v e r v i e w , C a n c e r Res., 3 0 ( 1 9 7 0 ) 5 5 9 - - 5 7 6 . 5 0 Miller, J . A . a n d E.C. Miller, C h e m i c a l c a r c i n o g e n e s i s , m e c h a n i s m a n d a p p r o a c h e s t o its c o n t r o l , J. Natl. C a n c e r Inst., 4 7 ( 1 9 7 1 ) v--xiv. 51 M o n t e s a n o , R . a n d H. B a r t s c h , M u t a g e n i c a n d c a r c i n o g e n i c N - n i t r o s o c o m p o u n d s : p o s s i b l e e n v i r o n m e n t a l h a z a r d s , M u t a t i o n R e s . , 1 9 7 6 (in press), 52 M o n t e s a n o , R. a n d L. T o m a t i s , (eds.) C h e m i c a l C a r c i n o g e n e s i s Essays, I A R C S c i e n t i f i c P u b l i c a t i o n s , No. 1 0 , L y o n , 1 9 7 4 . 53 M o n t e s a n o , R . , H. B a r t s c h a n d L. T o m a t i s , (eds.) S c r e e n i n g Tests i n C h e m i c a l C a r c i n o g e n e s i s , I A R C Scientific Publications, No. 12, Lyon, 1976. 5 4 N a t i o n a l A c a d e m y of S c i e n c e s , P a r t i c u l a r P o l y c y c l i c O r g a n i c M a t t e r , W a s h i n g t o n , D.C., 1 9 7 2 .
189
5 5 R a m e l , C., e d , R e p o r t of a s y m p o s i u m h e l d a t S k o k l o s t e r , S w e d e n , 1 9 7 2 , A m b i o S p e c i a l R e p o r t , No. 3, 1 9 7 3 . 5 6 R a n n u g , U., 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 m e i s t e r , T h e 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. 5 7 De S e r r e s , F . J . a n d W. S h e r i d a n , (eds.) T h e E v a l u a t i o n o f C h e m i c a l M u t a g e n i c i t y D a t a in R e l a t i o n t o P o p u l a t i o n R i s k , E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , No. 6 , 1 9 7 3 . 5 8 S t i c h , H . F . , R . H . C . S a n , J . A . Miller a n d E.C. Miller, V a r i o u s levels of D N A r e p a i r s y n t h e s i s i n X e r o d e r m a p i g m e n t o s i u m cells e x p o s e d t o t h e c a r c i n o g e n s N - h y d r o x y a n d N - a c e t o x y - 2 - a c e t y l a m i n o f l u o r e n e , N a t u r e N e w Biol., 2 3 8 ( 1 9 7 2 ) 9 - - 1 0 . 5 9 S t i e h , H . F . , R . H . C . S a n a n d Y. K a w a z o e , I n c r e a s e d s e n s i t i v i t y of X e r o d e r m a p i g m e n t o s u m cells t o s o m e c h e m i c a l c a r c i n o g e n s a n d m u t a g e n s , M u t a t i o n Res., 1 7 ( 1 9 7 3 ) 1 2 7 - - 1 3 7 . 6 0 S t o l t z , D . R . , L . A . Poirier, C.C. Irving, H . F . S t i c h , J . H . W e i s b u r g e r a n d H.C. Grice, E v a l u a t i o n o f s h o r t t e r m t e s t s f o r c a r c i n o g e n i c i t y , T o x i c o l . A p p l . P h a r m a c o l . , 29 ( 1 9 7 4 ) 1 5 7 - - 1 8 0 . 6 1 S u g i m u r a , T., T. Y a h a g i , M. N a g a o , M. T a k e u c h i , T. K a w a c h i , K. H a r a , E. Y a m a s a k i , T. M a t s u s h i m a , Y. H a s h i m o t o a n d M. O k a d a , V a l i d i t y o f m u t a g e n i c i t y t e s t s u s i n g m i c r o b e s as a r a p i d s c r e e n i n g m e t h o d f o r e n v i r o n m e n t a l c h e m i c a l s . In: R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s (eds.) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , L y o n , I A R C S c i e n t i f i c P u b l i c a t i o n s No. 1 2 ) 1 9 7 6 . 6 2 V a n D u u r e n , B . L . , B.M. G o l d s c h m i d t a n d I. S e i d m a n , C a r c i n o g e n i c a c t i v i t y o f di- a n d t r i f u n c t i o n a l ~c h l o r o e t h e r s a n d o f 1 , 4 - d i c h l o r o b u t e n e - 2 in I C R / H A Swiss m i c e , C a n c e r Res., 3 5 ( 1 9 7 5 ) 2 5 5 3 - - 2 5 5 7 . 6 3 V o g e l , E., T h e r e l a t i o n b e t w e e n m u t a t i o n a l p a t t e r n a n d c o n c e n t r a t i o n b y c h e m i c a l m u t a g e n s in D r o s o p h i l a , in R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s ( e d s . ) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , Lyon, IARC Scientific Publications No. 12), 1976. 64 WHO, Evaluation and Testing of Drugs for Mutagenicity: Principles and Problems, WHO Techn. Rep. Set., N o . 4 8 2 , 1 9 7 1 . 6 5 W H O , A s s e s s m e n t o f t h 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 e i t y of C h e m i c a l s , W H O T e e h n . R e p . Set., No. 546, 1974. 6 6 Zief, M. a n d C.H. S c h r a m m , C h l o r o e t h y l e n e o x i d e , C h e m . I n d . , ( 1 9 6 4 ) 6 6 0 - - - 6 6 1 . 6 7 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e Salmonella/microsome t e s t : A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. (Wash.) 7 2 ( 1 9 7 5 ) 5 1 3 5 - - 5 1 3 9 . 6 8 L o p r i e n o , N., R. B a r a l e , S. B a r o n c e l l i , C. B a u e r , G. B r o n z e t t i . A. C a m m e l l i n i , G. C e r c i g n a n i , C. Corsi, G. Gervasi, C. L e p o r i n i , R. Nieri, A . M . Rossi, G. S t r e t t i a n d G. T u r c h i , E v a l u a t i o n o f t h e g e n e t i c eff e c t s i n d u c e d b y v i n y l c h l o r i d e m o n o m e r ( V C M ) u n d e r m a m m a l i a n m e t a b o l i c a c t i v a t i o n : s t u d i e s in v i t r o a n d in vivo, M u t a t i o n Res., 4 0 ( 1 9 7 6 ) 85---96.
Mutation Research, 38 (1976) 177--190 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
PREDICTIVE VALUE OF MUTAGENICITY TESTS IN CHEMICAL CARCINOGENESIS
H. B A R T S C H International Agency for Research on Cancer, Unit o f Chemical Carcinogenesis, 150 cours Albert Thomas, 69008 Lyon, France
{Received October 10th, 1975) {Accepted November 28th, (1975)
An essential role of environmental factors in the aetiology of human cancer has been shown by the unequivocal evidence of the chemical origin of occupational cancer, as in the cases of urinary bladder tumours in workers exposed to aromatic amines [29,13,12] lung cancers in workers exposed to bis(chloromethyl) ether [30] or angiosarcomas of the liver in workers exposed to vinyl chloride [31] ; the well
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F i g . 2. L i v e r - e n z y m e - m e d i a t e d m u t a g e n i e i t y ( h i s + r e v e r t a n t c o l o n i e s o f S. t y p h i m u r i u m 1530/plate) of BD-VI rat tissue and individual human biopsy samples, represented by different characters. N-nitrosamine w e r e a s s a y e d a c c o r d i n g t o A m e s e t al. [ 3 ] , a n d v i n y l c h l o r i d e a s d e s c r i b e d b y B a r t s c h e t al. [ 7 ] . T h e c o n c e n t r a t i o n o f t h e c a r c i n o g e n s u s e d w a s w i t h i n a l i n e a r r a n g e o f t h e d o s e - r e s p o n s e c u r v e s o b t a i n e d i n a n assay system containin~ 9 000 ~ supernatant equivalent to 39 mg liver/plate. Number of spontaneous his + revertant colonies from appropriate controls (no co-factors or no substrate) was subtracted. Data from B a r t s c h e t al. [ 9 ] a n d u n p u b l i s h e d w o r k .
metabolites, but, as measured for individual Z, several carcinogens, which even belong to different chemical classes. This has been shown for five different compounds (Fig. 2). The identification of people who may be at exceptionally high risk for cancer because of a genetically determined enzyme profile may in future be of some help in establishing regulatory measures to limit the levels of carcinogens in the environment. Such levels should be established by taking into consideration the possible risk for the most susceptible individuals. The last part of this discussion concerns the usefulness of these mutagenicity tests in predicting the possible carcinogenic effects of chemicals in man. Despite the converging tendency of chemicals to be both carcinogenic and mutagenic, it is not known at present whether in future all carcinogens will be found to be mutagens and all mutagens carcinogenic. For the strong mutagens, such as base analogues, nitrous acid and hydroxylamine, no carcinogenic effect in animals has been reported so far [48]. These chemicals do not act via electrophilic intermediates. On the other hand, synthetic steroidal sex hormones, which are carcinogenic in animals [31], have not yet been reported to be mutagenic, and we have also been unable in our own laboratory to detect a mutagenic effect in tissue-mediated assays in vitro using the human and animal carcinogen a,a'-di-
185
ethylstilboestrol as substrate with the T A 1 0 0 strain of S. typhimurium (Bartsch, unpublished). These findings would suggest that for this class of compounds, a different cancer-inducing mechanism may be implied. There are various limitations of the mutagenicity test systems, since some of the factors that determine the processes of cancer development in vivo cannot be duplicated in, for example, mutagenicity systems in vitro. Some of the most important of these determining factors are the concentration of ultimate reactive metabolites available for reaction in organs and animal species with cellular macromolecules, which is a consequence of a critical balance between metabolic activation and detoxication processes; an organ-specific release of proximate or ultimate carcinogens by enzymic deconjugation; the biological half-life of metabolites; and organ-specific D N A repair or frequency of D N A replication in target or non-target organs and immuno-surveillance. The use of mutagenicity tests in the assessment of the carcinogenic risk due to chemicals justifies their availability, since, although an enormous number of new chemicals is entering the environment, the world capacity for testing for carcinogenicity is only about 500 compounds per year, and it is not possible to test all of them in long-term carcinogenicity tests [ 3 2 , 3 3 , 3 4 , 3 5 , 3 6 ] . It is necessary, therefore, to pre-screen in order to establish priorities for long-term testing. In evaluating these tests, four points should be emphasized: (1) positive results from mutagenicity tests given at level C {Table IV) cannot automatically be taken to imply a definite carcinogenic effect in man {level A); (2) positive or negative results from these tests cannot substitute for long-term carcinogenicity
TABLE
IV
FRAMEWORK
OF CARCINOGENICITY
Valid data o n
TEST PROCEDURES
Test s y s t e m
N o data o n
Carcinogenic in m a n
T h r e s h o l d dose; individual
T_ Target organ in man; high risk groups
Species and organ specificity d o s e response in animals
Mechanism o f m e t a b o l i c activation in animals and man; t y p e o f genetic damage
;
risk
A
Epidemiologicalstudies
l
Level
Positive
]
Predictive value for extrap o l a t i o n (at present limited); target organ; threshold d o s e
J
Careinogenieity test in animals I Positive
Mutagenicity tests Microbial, m a m m a l i a n , h u m a n cells/activation in vivo and in vitro Chemicals
Level
~ --
B
Species a n d / o r organ specificity correlation b e t w e e n mutagenic and carcinogenic p o t e n c y Level
C
;
186 testing in animals (level B); (3) the present methodology, in particular tissuemediated mutagenicity assays, is effective in predicting the carcinogenic potential of chemicals with a high probability, ranging at present between 80 and 90%, but gives no indication of target organs or species specificity of the carcinogenic action of the chemical; and, (4) the relative potency of a chemical as a mutagen cannot at present be correlated with its p o t e n c y as a carcinogen in experimental animals or man. Taking into account present reproducibility, cost, number of chemicals that can be examined in a short time and the scientific basis of the test, tissue-mediated mutagenicity procedures using well-characterized genetic indicators and a metabolically defined activation system in vitro (apart from the Drosophila test [63] ) appear at present to be the most promising short-term tests for the detection of adverse biological effects produced by chemicals. The present methodology still incurs the chance of false negative results, depending on the system used, either due to mutagen specificity, lack of appropriate cofactors for activation or to extreme reactivity and volatility of the c o m p o u n d or its metabolites [9]. However, the number of false negative results obtained in the tissue-mediated assay in vitro is very small in comparison with that obtained with other mutagenicity test procedures. As has often been mentioned, theoretical limitations are implied in the short-term tests currently used, because the development of cancer in vivo is determined by factors that cannot yet be duplicated in many of the mutagenicity test systems. Although there are still many problems involved in the interpretation of results of mutagenicity tests in terms of evaluating the carcinogenicity of chemicals, short-term tests can already be used in detecting possible cancer-causing agents with a sensitivity which did n o t exist ten years ago. They could thus be a powerful tool, when used in combination with epidemiological studies, in environmental control. This possibility, therefore, raises two important issues: (1) the validity of the short-term tests must be based on reliable corroboration by data from long-term tests in experimental animals; so, first of all the long-term data must be critically analysed and accepted to provide solid and definite background information; and, (2) the scientific community, as well as health authorities, must reach a concensus on the usefulness of results from these test systems in predicting possible carcinogenic effects of c o m p o u n d s in man. Otherwise, and if no public health measures are taken, for instance, to remove the agent from the environment or greatly to reduce exposure to it, cancer in man can only be treated and not prevented.
Acknowledgements
The author's experimental work was partially supported by the National Cancer Institute of the USA, Contract No. 1CP-55630. For valuable discussion and/or experimental assistance, the author is indebted to L. Tomatis, R. Montesano, C. Malaveille, A. Barbin, A. Camus, G. Planche, G. Brun and H. Br~sil. The help of Miss P. Stafford Smith and Mrs E. Ward in preparing the manuscript is gratefully acknowledged.
187
Note added in proof Loprieno et al. [68] have observed a pronounced mutagenic effect of vinyl chloride in Schizosaccharomyces pombe and Saccharomyces cerevisiae, only when mouse-liver microsomes were added to the assays in vitro. Vinyl chloride was also active in the host-mediated assay in mice. References 1 A m e s , B.N. a n d J. M c C a n n , 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 t e s t s y s t e m , in R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s (eds.) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , L y o n ( I A R C S c i e n t i f i c P u b lications No. 12), 1976. 2 A m e s , B.N., A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s in u r i n e : s t u d i e s w i t h t h e c a r c i n o g e n 2a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. (Wash.), 71 ( 1 9 7 4 ) 7 3 7 - - 7 4 1 . 3 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d D . F . 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 t e s t c o m b i n i n g liver h o m o g e n a t e s f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , P r o c . N a t l . A c a d . Sci. (Wash.), 7 0 (1973) 2281--2285. 4 B a r b i n , A., H. Bresil, A. C r o i s y , P. J a c q u i g n o n , C. Malaveille, R. M o n t e s a n o a n d H. B a r t s c h , IAvermicrosomeomediated formation of alkylating agents from vinyl bromide and vinyl chloride, Biochem. B i o p h y s . Res. C o m m u n . , 6 7 ( 1 9 7 5 ) 5 9 6 - - 6 0 3 . 5 B a r t s c h , H. a n d P.L. G r o v e r , C h e m i c a l c a r c i n o g e n e s i s a n d m u t a g e n e s i s , in T. S y m i n g t o n a n d R . L . Cart e r (eds.) S c i e n t i f i c F o u n d a t i o n s o f O n c o l o g y , 1 9 7 6 , p p . 3 3 4 - - 3 4 2 . 6 B a r t s c h , H. a n d R. 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