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Relationship between experimental results in mammals and man
Mutation Research, 31 (I975) 255-257 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
SHORT
255
COMMUNICATIONS
A review of the Committee 17 Report During the last few years the field of environmental mutagenesis has undergone a period of explosive development and the potentialities and practicalities of protecting the population against genetic hazards from chemicals are well known to most readers of this journal. The Council of the American Environmental Mutagen Society considered that the time was ripe for a more public statement and as a result of their initiative a report "Environmental Mutagenic Hazards" has now been published (Science, 187 (1975) 503-514.) being the product of a committee ("Committee 17" ) set up under the chairmanship of Dr. JOHN W. DRAKE. The report is aimed at those without a detailed knowledge of the field, particularly toxicologists, environmentalists and those involved with the work of regulatory agencies, and it is concerned only with nmtations in germinal cells and not with the possible effects of somatic mutations. The stated objectives of the report are to describe and discuss the test systems currently available for the detection of mutagenic activity, the evaluation and significance of the data obtained, the ways in which they may be applied to the protection of human populations, and the principles involved in regulatory procedures. By and large they have succeeded in these objectives and it is useful to have such a document at the present time. If some points are subject to criticism below, this is perhaps more for the benefit of the specialist but it may serve to show that not everything in the report is likely to be uncontentious. The introduction is brief, perhaps too brief. In considering the effects of mutations in populations there is, for example, no mention of the existence of heterozygous advantage of deleterious mutations. The statement that, in terms of human suffering, the summed effects of single gene mutations probably exceed the deleterious effects of changes in chromosome number or arrangements is also unlikely to meet with everyone's approval. Although there is general agreement that an increase in the mutation rate must inevitably lead to an increase in human suffering perhaps what is missing here is any indication that controversy exists among geneticists as to its magnitude.
Test systems The largest section in the report is given over to a summary and critical evaluation of the tests that are currently available for mutagenicity screening. One recommendation with which I take issue is that standard protocols are necessary to ensure high levels of reproducibility. They indeed help to achieve the latter but in doing so they may create a false sense of security. Unexpected differences with different protocols may both give important clues about the mode of action of a mutagen and point to the inadequacies of current protocols. The important thing about a testing protocol is that it should be sound, not standard. As any editor in this field knows, unsound protocols abound. Therefore recommendation of sound protocols is good, but reliance on these should not be a substitute for attempting to understand the problems posed by the properties and proposed usage of a given chemical and treating it appropriately. Is there such a thing as a protocol that will be ideal for all agents ? In dealing with chromosomal damage the report rightly emphasises the point
250
REVIEW
(often seemingly ignored) that the induction of chromosomal breaks is not a valid index of heritable damage. Rather it should be regarded as a warning signal which should lead to a more extensive screening for rearrangements. It is now clear that m a n y mutagens (and carcinogens) need metabolic activation within the mammalian body and the report describes ways in which metabolic activation m a y be incorporated into tests involving cellular mutation systems. I feel, however, that our understanding of metabolic activation falls short of what one is led to believe. We do not really have enough experience to give us confidence that current techniques can detect all types of activation. For example, the well-known activated mutagen dimethyl nitrosamine does not work in the commonly used soft agarmicrosome plate test unless exquisitely sensitive bacteria are used, although it readily mutates wild-type bacteria if the reaction is carried out in the test-tube without soft agar. This is not, of course, an argument for failing to use available methods, merely an indication that further improvements are desirable. The report goes on to make the good points that any detailed examination of a compound should include an examination of its metabolic fates (at least in experimental animals), and that this should involve sufficiently large numbers of genetically different individuals to make likely the discernment of genetically determined variability in metabolic activities. I was pleased to see that the vexed question of the existence of thresholds ("noeffect levels") is one on which the authors have felt able to take a sound and uncompromising position. They state that it is usually impossible to establish with confidence that sufficient statistical accuracy has been achieved to conclude that a nmtagenic effect is truly absent. In the absence of other evidence, therefore, good practice demands that a linear interpolation be made between the spontaneous rate at zero dose and the response for the lowest dose at which reliable data exist. Extrapolation to man
The identification of the nmtagenic activity of a substance is only tile first step in the evaluation of its possible hazard to man. One needs to know about production levels and distribution patterns (existing or proposed), about persistence, metabolic disposition in man, genetically significant concentrations and individual variability. All these aspects are covered, albeit briefly, in the report. The use of a radiation equivalent dose to quantify and compare the effects of different chemicals has been suggested elsewhere and the report endorses it as a possible approach. In an appendix there are attempts to estimate the nmtagenicity of hycanthone methanesulphonate, ethyl methanesulphonate, and nitrite in terms of radiation equivalents. The most surprising feature of these is the close similarity of the radiation equivalent doses using different genetic end-points. For hycanthone, for example, 3 mg/kg (the human therapeutic dose) is estimated to be equivalent to 1.2 to 2. 4 rad (specific locus mutations in mice), about I rad (chromosome aberrations in cultured human somatic cells) and 1.8 rad (sex-linked recessive lethal mutations in Drosophila). Perhaps even more unexpected is the fact that with ethyl methanesulphonate, which is believed to cause mutations in cellular systems by misreplication of guanines alkylated in the 0-6 position, the estimation based on mouse dominant lethal and translocation mutations (O.0I M _~ 16oo rad) is the same as that based on mouse specific locus mutations (o.oi M ---=15oo-2ooo rad). One interpretation of these results might be that the mouse specific locus test does not detect mutations resulting
REVIEW
257
from single base misreplications and that both it and the tests for chromosomal damage are detecting damage due to a different mechanism. Certainly it seems likely that as more data become available radiation equivalent doses will begin to reveal the specificities of different mutagens and these specificities must needs be taken into account in the evaluation of human hazards.
Risks and benefits Ultimately, of course, when all the information has been gathered, it is necessary to make a risk-benefit evaluation. The 1972 B E I R Report is quoted with apparent approval for estimating the future cost of human exposure to ionizing radiation in terms of health costs paid in present dollars (between $12 and $12o per man-rem) and the extension of this approach to chemical mutagens is recommended. There are, however, many who, while conceding that monerary considerations are important, would wish to take account of other factors such as the intensity of the human suffering and the foreseeable level of non-genetic ill health likely to obtain in the descendants of the exposed population. In considering the overall risk from environmental mutagens the report is firmly in favour of a single maximum budget of 5 radiation equivalents per man-reproductive period to include ionizing radiations as well as chemicals. They furthermore recommend that no single mutagenic agent should be allowed to exceed lO% of this budget. Since I also suggested some years ago a single budget to include the effects of radiations and chemicals (and a combined Environmental Genetic Hazards Commission to regulate it) it has been pointed out to me more than once that this is not necessarily a logical step. In comparing the risks and benefits of a given agent it is irrelevant what other risks and benefits are already accepted for other agents provided the new risks (or benefits) do not interact with those already in existence (and there is little likelihood of evidence on this point being available for mutational risks). This argument has some force, but I can yet see many practical advantages in a single maximum budget with a common supervisory body. Such a move would, of course, detract from the powers of independent bodies at present responsible for regulating the safety of such various agents as drugs, pesticides, food additives, cosmetics etc., and might not meet with their approval. In conclusion, I recommend the Committee 17 Report to all interested parties. Do not, as a busy man is sometimes tempted to do with a fashionable book or play, accept a review as a substitute for the real thing. There are many points raised in the report, only a few of which have been discussed here, which deserve thoughtful consideration. Some of them would undoubtedly profit from open discussion in the columns of this Journal.
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BNr 9QG (Great Britain) Received January 3Ist, 1975
B. A. BRIDGES
SHORT
255
COMMUNICATIONS
A review of the Committee 17 Report During the last few years the field of environmental mutagenesis has undergone a period of explosive development and the potentialities and practicalities of protecting the population against genetic hazards from chemicals are well known to most readers of this journal. The Council of the American Environmental Mutagen Society considered that the time was ripe for a more public statement and as a result of their initiative a report "Environmental Mutagenic Hazards" has now been published (Science, 187 (1975) 503-514.) being the product of a committee ("Committee 17" ) set up under the chairmanship of Dr. JOHN W. DRAKE. The report is aimed at those without a detailed knowledge of the field, particularly toxicologists, environmentalists and those involved with the work of regulatory agencies, and it is concerned only with nmtations in germinal cells and not with the possible effects of somatic mutations. The stated objectives of the report are to describe and discuss the test systems currently available for the detection of mutagenic activity, the evaluation and significance of the data obtained, the ways in which they may be applied to the protection of human populations, and the principles involved in regulatory procedures. By and large they have succeeded in these objectives and it is useful to have such a document at the present time. If some points are subject to criticism below, this is perhaps more for the benefit of the specialist but it may serve to show that not everything in the report is likely to be uncontentious. The introduction is brief, perhaps too brief. In considering the effects of mutations in populations there is, for example, no mention of the existence of heterozygous advantage of deleterious mutations. The statement that, in terms of human suffering, the summed effects of single gene mutations probably exceed the deleterious effects of changes in chromosome number or arrangements is also unlikely to meet with everyone's approval. Although there is general agreement that an increase in the mutation rate must inevitably lead to an increase in human suffering perhaps what is missing here is any indication that controversy exists among geneticists as to its magnitude.
Test systems The largest section in the report is given over to a summary and critical evaluation of the tests that are currently available for mutagenicity screening. One recommendation with which I take issue is that standard protocols are necessary to ensure high levels of reproducibility. They indeed help to achieve the latter but in doing so they may create a false sense of security. Unexpected differences with different protocols may both give important clues about the mode of action of a mutagen and point to the inadequacies of current protocols. The important thing about a testing protocol is that it should be sound, not standard. As any editor in this field knows, unsound protocols abound. Therefore recommendation of sound protocols is good, but reliance on these should not be a substitute for attempting to understand the problems posed by the properties and proposed usage of a given chemical and treating it appropriately. Is there such a thing as a protocol that will be ideal for all agents ? In dealing with chromosomal damage the report rightly emphasises the point
250
REVIEW
(often seemingly ignored) that the induction of chromosomal breaks is not a valid index of heritable damage. Rather it should be regarded as a warning signal which should lead to a more extensive screening for rearrangements. It is now clear that m a n y mutagens (and carcinogens) need metabolic activation within the mammalian body and the report describes ways in which metabolic activation m a y be incorporated into tests involving cellular mutation systems. I feel, however, that our understanding of metabolic activation falls short of what one is led to believe. We do not really have enough experience to give us confidence that current techniques can detect all types of activation. For example, the well-known activated mutagen dimethyl nitrosamine does not work in the commonly used soft agarmicrosome plate test unless exquisitely sensitive bacteria are used, although it readily mutates wild-type bacteria if the reaction is carried out in the test-tube without soft agar. This is not, of course, an argument for failing to use available methods, merely an indication that further improvements are desirable. The report goes on to make the good points that any detailed examination of a compound should include an examination of its metabolic fates (at least in experimental animals), and that this should involve sufficiently large numbers of genetically different individuals to make likely the discernment of genetically determined variability in metabolic activities. I was pleased to see that the vexed question of the existence of thresholds ("noeffect levels") is one on which the authors have felt able to take a sound and uncompromising position. They state that it is usually impossible to establish with confidence that sufficient statistical accuracy has been achieved to conclude that a nmtagenic effect is truly absent. In the absence of other evidence, therefore, good practice demands that a linear interpolation be made between the spontaneous rate at zero dose and the response for the lowest dose at which reliable data exist. Extrapolation to man
The identification of the nmtagenic activity of a substance is only tile first step in the evaluation of its possible hazard to man. One needs to know about production levels and distribution patterns (existing or proposed), about persistence, metabolic disposition in man, genetically significant concentrations and individual variability. All these aspects are covered, albeit briefly, in the report. The use of a radiation equivalent dose to quantify and compare the effects of different chemicals has been suggested elsewhere and the report endorses it as a possible approach. In an appendix there are attempts to estimate the nmtagenicity of hycanthone methanesulphonate, ethyl methanesulphonate, and nitrite in terms of radiation equivalents. The most surprising feature of these is the close similarity of the radiation equivalent doses using different genetic end-points. For hycanthone, for example, 3 mg/kg (the human therapeutic dose) is estimated to be equivalent to 1.2 to 2. 4 rad (specific locus mutations in mice), about I rad (chromosome aberrations in cultured human somatic cells) and 1.8 rad (sex-linked recessive lethal mutations in Drosophila). Perhaps even more unexpected is the fact that with ethyl methanesulphonate, which is believed to cause mutations in cellular systems by misreplication of guanines alkylated in the 0-6 position, the estimation based on mouse dominant lethal and translocation mutations (O.0I M _~ 16oo rad) is the same as that based on mouse specific locus mutations (o.oi M ---=15oo-2ooo rad). One interpretation of these results might be that the mouse specific locus test does not detect mutations resulting
REVIEW
257
from single base misreplications and that both it and the tests for chromosomal damage are detecting damage due to a different mechanism. Certainly it seems likely that as more data become available radiation equivalent doses will begin to reveal the specificities of different mutagens and these specificities must needs be taken into account in the evaluation of human hazards.
Risks and benefits Ultimately, of course, when all the information has been gathered, it is necessary to make a risk-benefit evaluation. The 1972 B E I R Report is quoted with apparent approval for estimating the future cost of human exposure to ionizing radiation in terms of health costs paid in present dollars (between $12 and $12o per man-rem) and the extension of this approach to chemical mutagens is recommended. There are, however, many who, while conceding that monerary considerations are important, would wish to take account of other factors such as the intensity of the human suffering and the foreseeable level of non-genetic ill health likely to obtain in the descendants of the exposed population. In considering the overall risk from environmental mutagens the report is firmly in favour of a single maximum budget of 5 radiation equivalents per man-reproductive period to include ionizing radiations as well as chemicals. They furthermore recommend that no single mutagenic agent should be allowed to exceed lO% of this budget. Since I also suggested some years ago a single budget to include the effects of radiations and chemicals (and a combined Environmental Genetic Hazards Commission to regulate it) it has been pointed out to me more than once that this is not necessarily a logical step. In comparing the risks and benefits of a given agent it is irrelevant what other risks and benefits are already accepted for other agents provided the new risks (or benefits) do not interact with those already in existence (and there is little likelihood of evidence on this point being available for mutational risks). This argument has some force, but I can yet see many practical advantages in a single maximum budget with a common supervisory body. Such a move would, of course, detract from the powers of independent bodies at present responsible for regulating the safety of such various agents as drugs, pesticides, food additives, cosmetics etc., and might not meet with their approval. In conclusion, I recommend the Committee 17 Report to all interested parties. Do not, as a busy man is sometimes tempted to do with a fashionable book or play, accept a review as a substitute for the real thing. There are many points raised in the report, only a few of which have been discussed here, which deserve thoughtful consideration. Some of them would undoubtedly profit from open discussion in the columns of this Journal.
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BNr 9QG (Great Britain) Received January 3Ist, 1975
B. A. BRIDGES