Ann. occup. Hjy. Vol. 20. pp. 297-301. Pergaroon Pna 1977. Printed in Great Britain
THE SELECTION OF APPROPRIATE CHEMICAL CLASS CONTROLS FOR USE WITH SHORT-TERM TESTS FOR POTENTIAL CARCINOGENICITY JOHN ASHBY and I. F. H. PURCHASE
Abstract—The approach described below allows short-term test predictions of carcinogenkity to be extrapolated to the in vivo situation with confidence for some compounds, while for other compounds it leads to serious doubts about the in vivo significance of the prediction.
of relatively reliable and rapid short-term tests for detecting animal carcinogens has provided a major new technique for detecting potential carcinogens in the environment or the workplace (AMES et al., 1975; MCCANN and AMES, 1976; PURCHASE etal, 1976; 1977 in preparation). However, experience has shown that all of the currently available tests are liable to make false predictions of in vivo carcinogenicity. False positive predictions could result in unnecessary political or commercial pressures on a chemical or product. Likewise, false negative predictions could result in a false sense of security. There is already evidence to suggest that false negative predictions may be a significant problem and that they will be selectively generated within specific classes of chemicals (PURCHASE et al., 1976; 1977 in preparation; ASHBY et al., to be published). There is, therefore, a need to develop an approach to short-term testing which will enable the likely in vivo significance of a test result to be assessed at the time of testing. The approach to this problem which is outlined below enables the short-term test predictions for some compounds to be extrapolated to the in vivo situation with confidence, whilst for other compounds it leads to serious doubts about the in vivo significance of the prediction. The basis of this approach lies in using the large quantity of information concerning which chemicals can induce cancer in animals, which has been built up over the past fifty years (bibliography). This information can be used in two distinct ways. The first is for rapid identification of the most likely potential carcinogens within a group of untested compounds before any testing takes place. This is illustrated by compound II in Fig. 1. As it is the methyl analogue of yS-naphthylamine (compound I, Fig. 1) which is a well known animal and human carcinogen (bibliography), one would have few reservations in classifying it as a potential carcinogen in advance of any in vivo testing. The second use of historical data can be illustrated by the same example. If compound II was known to be an environmentally or industrially important chemical it would clearly be necessary at least to evaluate its potential carcinogenicity in an in vitro short-term test. However, if any credence is to be placed on the results of the THE ADVENT
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Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
Imperial Chemical Industries Limited, Central Toxicology Laboratory, Alderley Park, Cheshire
298
JOHN ASHBV and I. F. H. PURCHASE
test chosen, it must be one which can clearly demonstrate its ability to detect a structurally related carcinogen as positive and a structurally related non-carcinogen as negative. In the present example, therefore, /3-naphthylamine (I) should act as the positive control and a-naphthylamine (III) as the negative control. Should the test fail to detect /?-naphthylamine as positive then very little reliance could be placed on a negative finding for compound II. Conversely, if the test were to find a-naphthylamine positive then the likely in vivo significance of a positive test response for compound II would be questionable. Both a-naphthylamine and /?-naphthylamine have been evaluated in the Ames test (MCCANN and AMES, 1976; PURCHASE et al., 1977) and in the cell transformation test of Styles (STYLES, to be published; PURCHASE et al., 1976; 1977 in preparation) and only the Ames test was able to differentiate correctly between them. This test would, therefore, be the most appropriate one with which to evaluate compound II for potential carcinogenicity. An example with a different outcome is illustrated by consideration of the carcinogen butter yellow (compound IV, Fig. 1) (bibliography). This compound has a closely related (structurally) non-carcinogenic analogue, 3-methyl-4-dimethylaminoazobenzene (compound V, Fig. 1) (bibliography). Therefore, before evaluating any hitherto untested compounds which may be structurally related to butter yellow (and therefore potentially carcinogenic) the performance of the available short-term tests with these two compounds should be evaluated. Both compounds have, in fact, been submitted to the Ames test and the cell transformation test of STYLES (to be published). Butter yellow gave a negative result in the Ames test using the plate incorporation assay (AMES et al., 1973), and has given a negative Ames test response on all but one occasion in this laboratory (PURCHASE et al., 1976; 1977 in preparation; D. ANDERSON, 1977, personal communication). This test is therefore not the preferred test for evaluating structural analogues of butter yellow for potential carcinogenicity. In contrast, the cell transformation test of Styles finds butter yellow positive and its non-carcinogenic analogue (IV) negative (Fig. 2) (STYLES, 1977, personal communication and permission to reproduce unpublished material. For a detailed description of this test and the interpretation of Fig. 2, see PURCHASE et al., 1976 and STYLES, to be published). This test therefore appears to be consistently reflecting the true in vivo
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
FIG. 1. Structural formulae. I /3-naphthylamine; II methyl analogue of I; III a-naphthylamine; IV butter yellow; V 3-methyl-4-dimethylaminoazobenzene.
Chemical class controls for use with short-term tests for potential carcinogenicity
299
o
I
100 0 025 0.25 Z5 25 Concentration, fj.g/m\
250
Fio. 2. Cell survival and transformation curves given by butter yellow ( — • — • — ; positive) and its 3-methyl analogue (—A—A—; negative) in the cell transformation assay of Styles.
carcinogenic properties of such compounds and is consequently the preferred test to use when evaluating structural analogues of butter yellow for potential carcinogenicity. It is not sufficient, however, for a test to establish its suitability for testing a given class of compounds on a single occasion. There is sufficient variability in the test responses for some compounds (ASHBY et al., to be published), due probably to variations in culture medium, microsome enzyme levels etc., to demand that structurally relevant controls be incorporated into each short-term test experiment. This can be illustrated by a recent experience in this laboratory. It has been reported (PURCHASE etal., 1976; 1977 in preparation; MCCANN and AMES, 1976) that the Ames test can detect the carcinogen hydrazine as positive. A series of hitherto untested hydrazine derivatives has recently been evaluated in the Ames test to determine if they had carcinogenic potential. The results were all negative, but so also was hydrazine and it was therefore decided to abandon the study (E. LONGSTAFF and J. ASHBY, 1976, unpublished results). However, had hydrazine not been tested as the chemical class positive control the hydrazine analogues would probably have been cleared of potential carcinogenicity. This would have been premature and might have been wrong. In conclusion, two points emerge. First, there is a need for those who are testing new compounds in any of the available short-term test systems to acquire a collection of structurally related carcinogens and non-carcinogens for the classes of compounds they are likely to be testing. These can be denned and synthesised by reference to the carcinogenicity literature (bibliography). Second, when short-term test positive responses are obtained for compounds of a chemical class which has not previously been associated with the chemical induction of cancer, the available long-term testing facilities should be applied to the definition of carcinogens and non-carcinogens within that class (in such cases the short-term test experiments would have been
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
E £ 300 •ft
300
JOHN ASHBY and I. F. H. PURCHASE
arbitrarily controlled with a known carcinogen and mutagen, e.g. 2-acetylaminofluorene). Until our collective experience of short-term tests has developed further it is important to continue to accept that some classes of carcinogens will always be negative in at least some of the available short-term tests and that the positive predictions given for some classes of compounds will always fail to anticipate the eventual in vivo outcome correctly. Efforts should be made to define and either accept or rectify these test short-comings in order that short-term tests can realise their full predictive potential.
AMES, B. N., MCCANN, J. and YAMASAKI, E. (1975) Mutat. Res. 31, 347. ASHBY, J., STYLES, J. A. and ANDERSON, D. (1977) Br. J. Cancer, to be published.
MCCANN, J. and AMES, B. N. (1976) Proc. natn Acad. Sci. U.S.A. 73, 950. PURCHASE, I. F. H., LONGSTAFF, E., ASHBY, J., STYLES, J. A., ANDERSON, D., LEFEVRE, P. A. and
WESTWOOD, F. R. (1976) Nature, Lond. 264, 624. STYLES, J. A. (1977) Br. J. Cancer, to be published. BIBLIOGRAPHY Survey of Compounds which have been tested for Carcinogenic Activity. U.S. Public Health Service Publication No. 149 and Supplements. U.S. Government Printing Office, Washington D.C. NATIONAL INSTITUTE OF OCCUPATIONAL SAFETY AND HEALTH (1975). NIOSH List of Suspected
Carcinogens. Fed. Reg. 40, 26390. SAX, N. I. (1975) Dangerous Properties of Industrial Materials. 4th Ed. Van Nostrand, Wokingham, Berks. HOWE, J. R. (1975) Lab. Pract. 457-467. Contains a useful bibliography of related material. HUEPER, W. C. (1975) Archs ind. Hlth 11, 494-504. Contains a general bibliography up to 1955. THE WORLD HEALTH ORGANISATION in collaboration with the International Association for Research on Cancer, have produced an ongoing series of monographs in which the carcinogenicity hazard to man of a large variety of compounds is assessed. IARC, Lyon, France. ARCOS, J. E., ARGUS, M. F. and WOLF, G. Chemical Induction of Cancer, Vol. I (1968); Vol. Ila and
lib (1974). Academic Press, New York. DISCUSSION J. R. GLOVER (Welsh National School of Medicine): Have you been able to go back to the simple carcinogens, such as /S-naphthylamine, o-tolidine and benzidine, which are very useful starting compounds in the chemical industry, and render them biologically harmless, yet still chemically active by using your predictive knowledge? DR ASHBY: This is certainly possible and we have, in fact, achieved it on several occasions. A good example is the inactivation of benzidine as a carcinogen by the placement of methyl groups on all of the ortho-positions. J. BLOW (ITT Medical Services): At a meeting of the Royal Society of Medicine last week we were told that trichloroethylene produces cancer in animals. If the short term carcinogenicity tests are also positive what advice would you offer to those of us in industry who use large quantities of trichloroethylene? DR ASHBY: The important point here is the validity of the animal in vivo data. If this is generally agreed upon then adequate precautions should be considered for the safe handling of this material. The addition of positive short-term test data would merely strengthen ones belief in the in vivo data. At the moment, halocarbons in general are presenting short-term tests with problems. It is an area where there appear to be substantial differences between short-term and long-term data. A. MUNN (Monsanto Europe): Surely this situation calls for epidemiological studies 7 As Alexander Pope said: "The proper study of mankind is man". DR ASHBY: Certainly. This should form the basis for deciding what changes, if any, in handling procedures are called for. The proper study of the future, in such cases, may in fact be the past.
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
REFERENCES AMES, B. N., DURSTON, W. E., YAMASAKI, E. and LEE, F. D. (1973) Proc. natn Acad. Sci., U.S.A. 70, 2281.
Chemical class controls for use with short-term tests for potential carcinogenicity
301
B. HARVEY (University of Aston): If epidemiology produces negative results, this is useful; if the results are positive isn't that when you should look for alternative exposures? DR ASHBY: I agree.
K. S. WILLIAMSON (ICI Ltd): Have we information on the predictivity of animal tests in the detection of human carcinogens ? You have demonstrated in a most interesting way the predictivity of the short-term tests in relation to animal tests. It is my impression that there is no similar information on the relationship of a test in one animal species to another species or to man. I. F. H. PURCHASE (Central Toxicology Laboratory, ICI Ltd): Using the NCI Compounds which have been Tested for Carcinogenecity as a basis, we have found that only 80% of carcinogens in one species are also carcinogenic in a second species.
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
THE SELECTION OF APPROPRIATE CHEMICAL CLASS CONTROLS FOR USE WITH SHORT-TERM TESTS FOR POTENTIAL CARCINOGENICITY JOHN ASHBY and I. F. H. PURCHASE
Abstract—The approach described below allows short-term test predictions of carcinogenkity to be extrapolated to the in vivo situation with confidence for some compounds, while for other compounds it leads to serious doubts about the in vivo significance of the prediction.
of relatively reliable and rapid short-term tests for detecting animal carcinogens has provided a major new technique for detecting potential carcinogens in the environment or the workplace (AMES et al., 1975; MCCANN and AMES, 1976; PURCHASE etal, 1976; 1977 in preparation). However, experience has shown that all of the currently available tests are liable to make false predictions of in vivo carcinogenicity. False positive predictions could result in unnecessary political or commercial pressures on a chemical or product. Likewise, false negative predictions could result in a false sense of security. There is already evidence to suggest that false negative predictions may be a significant problem and that they will be selectively generated within specific classes of chemicals (PURCHASE et al., 1976; 1977 in preparation; ASHBY et al., to be published). There is, therefore, a need to develop an approach to short-term testing which will enable the likely in vivo significance of a test result to be assessed at the time of testing. The approach to this problem which is outlined below enables the short-term test predictions for some compounds to be extrapolated to the in vivo situation with confidence, whilst for other compounds it leads to serious doubts about the in vivo significance of the prediction. The basis of this approach lies in using the large quantity of information concerning which chemicals can induce cancer in animals, which has been built up over the past fifty years (bibliography). This information can be used in two distinct ways. The first is for rapid identification of the most likely potential carcinogens within a group of untested compounds before any testing takes place. This is illustrated by compound II in Fig. 1. As it is the methyl analogue of yS-naphthylamine (compound I, Fig. 1) which is a well known animal and human carcinogen (bibliography), one would have few reservations in classifying it as a potential carcinogen in advance of any in vivo testing. The second use of historical data can be illustrated by the same example. If compound II was known to be an environmentally or industrially important chemical it would clearly be necessary at least to evaluate its potential carcinogenicity in an in vitro short-term test. However, if any credence is to be placed on the results of the THE ADVENT
297
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
Imperial Chemical Industries Limited, Central Toxicology Laboratory, Alderley Park, Cheshire
298
JOHN ASHBV and I. F. H. PURCHASE
test chosen, it must be one which can clearly demonstrate its ability to detect a structurally related carcinogen as positive and a structurally related non-carcinogen as negative. In the present example, therefore, /3-naphthylamine (I) should act as the positive control and a-naphthylamine (III) as the negative control. Should the test fail to detect /?-naphthylamine as positive then very little reliance could be placed on a negative finding for compound II. Conversely, if the test were to find a-naphthylamine positive then the likely in vivo significance of a positive test response for compound II would be questionable. Both a-naphthylamine and /?-naphthylamine have been evaluated in the Ames test (MCCANN and AMES, 1976; PURCHASE et al., 1977) and in the cell transformation test of Styles (STYLES, to be published; PURCHASE et al., 1976; 1977 in preparation) and only the Ames test was able to differentiate correctly between them. This test would, therefore, be the most appropriate one with which to evaluate compound II for potential carcinogenicity. An example with a different outcome is illustrated by consideration of the carcinogen butter yellow (compound IV, Fig. 1) (bibliography). This compound has a closely related (structurally) non-carcinogenic analogue, 3-methyl-4-dimethylaminoazobenzene (compound V, Fig. 1) (bibliography). Therefore, before evaluating any hitherto untested compounds which may be structurally related to butter yellow (and therefore potentially carcinogenic) the performance of the available short-term tests with these two compounds should be evaluated. Both compounds have, in fact, been submitted to the Ames test and the cell transformation test of STYLES (to be published). Butter yellow gave a negative result in the Ames test using the plate incorporation assay (AMES et al., 1973), and has given a negative Ames test response on all but one occasion in this laboratory (PURCHASE et al., 1976; 1977 in preparation; D. ANDERSON, 1977, personal communication). This test is therefore not the preferred test for evaluating structural analogues of butter yellow for potential carcinogenicity. In contrast, the cell transformation test of Styles finds butter yellow positive and its non-carcinogenic analogue (IV) negative (Fig. 2) (STYLES, 1977, personal communication and permission to reproduce unpublished material. For a detailed description of this test and the interpretation of Fig. 2, see PURCHASE et al., 1976 and STYLES, to be published). This test therefore appears to be consistently reflecting the true in vivo
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
FIG. 1. Structural formulae. I /3-naphthylamine; II methyl analogue of I; III a-naphthylamine; IV butter yellow; V 3-methyl-4-dimethylaminoazobenzene.
Chemical class controls for use with short-term tests for potential carcinogenicity
299
o
I
100 0 025 0.25 Z5 25 Concentration, fj.g/m\
250
Fio. 2. Cell survival and transformation curves given by butter yellow ( — • — • — ; positive) and its 3-methyl analogue (—A—A—; negative) in the cell transformation assay of Styles.
carcinogenic properties of such compounds and is consequently the preferred test to use when evaluating structural analogues of butter yellow for potential carcinogenicity. It is not sufficient, however, for a test to establish its suitability for testing a given class of compounds on a single occasion. There is sufficient variability in the test responses for some compounds (ASHBY et al., to be published), due probably to variations in culture medium, microsome enzyme levels etc., to demand that structurally relevant controls be incorporated into each short-term test experiment. This can be illustrated by a recent experience in this laboratory. It has been reported (PURCHASE etal., 1976; 1977 in preparation; MCCANN and AMES, 1976) that the Ames test can detect the carcinogen hydrazine as positive. A series of hitherto untested hydrazine derivatives has recently been evaluated in the Ames test to determine if they had carcinogenic potential. The results were all negative, but so also was hydrazine and it was therefore decided to abandon the study (E. LONGSTAFF and J. ASHBY, 1976, unpublished results). However, had hydrazine not been tested as the chemical class positive control the hydrazine analogues would probably have been cleared of potential carcinogenicity. This would have been premature and might have been wrong. In conclusion, two points emerge. First, there is a need for those who are testing new compounds in any of the available short-term test systems to acquire a collection of structurally related carcinogens and non-carcinogens for the classes of compounds they are likely to be testing. These can be denned and synthesised by reference to the carcinogenicity literature (bibliography). Second, when short-term test positive responses are obtained for compounds of a chemical class which has not previously been associated with the chemical induction of cancer, the available long-term testing facilities should be applied to the definition of carcinogens and non-carcinogens within that class (in such cases the short-term test experiments would have been
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
E £ 300 •ft
300
JOHN ASHBY and I. F. H. PURCHASE
arbitrarily controlled with a known carcinogen and mutagen, e.g. 2-acetylaminofluorene). Until our collective experience of short-term tests has developed further it is important to continue to accept that some classes of carcinogens will always be negative in at least some of the available short-term tests and that the positive predictions given for some classes of compounds will always fail to anticipate the eventual in vivo outcome correctly. Efforts should be made to define and either accept or rectify these test short-comings in order that short-term tests can realise their full predictive potential.
AMES, B. N., MCCANN, J. and YAMASAKI, E. (1975) Mutat. Res. 31, 347. ASHBY, J., STYLES, J. A. and ANDERSON, D. (1977) Br. J. Cancer, to be published.
MCCANN, J. and AMES, B. N. (1976) Proc. natn Acad. Sci. U.S.A. 73, 950. PURCHASE, I. F. H., LONGSTAFF, E., ASHBY, J., STYLES, J. A., ANDERSON, D., LEFEVRE, P. A. and
WESTWOOD, F. R. (1976) Nature, Lond. 264, 624. STYLES, J. A. (1977) Br. J. Cancer, to be published. BIBLIOGRAPHY Survey of Compounds which have been tested for Carcinogenic Activity. U.S. Public Health Service Publication No. 149 and Supplements. U.S. Government Printing Office, Washington D.C. NATIONAL INSTITUTE OF OCCUPATIONAL SAFETY AND HEALTH (1975). NIOSH List of Suspected
Carcinogens. Fed. Reg. 40, 26390. SAX, N. I. (1975) Dangerous Properties of Industrial Materials. 4th Ed. Van Nostrand, Wokingham, Berks. HOWE, J. R. (1975) Lab. Pract. 457-467. Contains a useful bibliography of related material. HUEPER, W. C. (1975) Archs ind. Hlth 11, 494-504. Contains a general bibliography up to 1955. THE WORLD HEALTH ORGANISATION in collaboration with the International Association for Research on Cancer, have produced an ongoing series of monographs in which the carcinogenicity hazard to man of a large variety of compounds is assessed. IARC, Lyon, France. ARCOS, J. E., ARGUS, M. F. and WOLF, G. Chemical Induction of Cancer, Vol. I (1968); Vol. Ila and
lib (1974). Academic Press, New York. DISCUSSION J. R. GLOVER (Welsh National School of Medicine): Have you been able to go back to the simple carcinogens, such as /S-naphthylamine, o-tolidine and benzidine, which are very useful starting compounds in the chemical industry, and render them biologically harmless, yet still chemically active by using your predictive knowledge? DR ASHBY: This is certainly possible and we have, in fact, achieved it on several occasions. A good example is the inactivation of benzidine as a carcinogen by the placement of methyl groups on all of the ortho-positions. J. BLOW (ITT Medical Services): At a meeting of the Royal Society of Medicine last week we were told that trichloroethylene produces cancer in animals. If the short term carcinogenicity tests are also positive what advice would you offer to those of us in industry who use large quantities of trichloroethylene? DR ASHBY: The important point here is the validity of the animal in vivo data. If this is generally agreed upon then adequate precautions should be considered for the safe handling of this material. The addition of positive short-term test data would merely strengthen ones belief in the in vivo data. At the moment, halocarbons in general are presenting short-term tests with problems. It is an area where there appear to be substantial differences between short-term and long-term data. A. MUNN (Monsanto Europe): Surely this situation calls for epidemiological studies 7 As Alexander Pope said: "The proper study of mankind is man". DR ASHBY: Certainly. This should form the basis for deciding what changes, if any, in handling procedures are called for. The proper study of the future, in such cases, may in fact be the past.
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
REFERENCES AMES, B. N., DURSTON, W. E., YAMASAKI, E. and LEE, F. D. (1973) Proc. natn Acad. Sci., U.S.A. 70, 2281.
Chemical class controls for use with short-term tests for potential carcinogenicity
301
B. HARVEY (University of Aston): If epidemiology produces negative results, this is useful; if the results are positive isn't that when you should look for alternative exposures? DR ASHBY: I agree.
K. S. WILLIAMSON (ICI Ltd): Have we information on the predictivity of animal tests in the detection of human carcinogens ? You have demonstrated in a most interesting way the predictivity of the short-term tests in relation to animal tests. It is my impression that there is no similar information on the relationship of a test in one animal species to another species or to man. I. F. H. PURCHASE (Central Toxicology Laboratory, ICI Ltd): Using the NCI Compounds which have been Tested for Carcinogenecity as a basis, we have found that only 80% of carcinogens in one species are also carcinogenic in a second species.
Downloaded from http://annhyg.oxfordjournals.org/ at University of Victoria on July 11, 2015
