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Assessment and evaluation of genotoxicity findings
Mutation Research, 291 (1993) 87-91
87
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1161/93/$06.00
MUTENV 08860
A s s e s s m e n t and evaluation of genotoxicity findings * Stephan Madle
a
and Rainer Lang b
a Federal Health Office, Max-uon-Pettenkofer-lnstitute, Berlin, Germany and b Schering AG, Experimental Toxicology, Berlin, Germany (Received 20 October 1992) (Accepted 21 October 1992)
Keywords: Interpretation of individual genotoxicity findings; Assessment of genotoxicity data; Evaluation of genotoxicity data
It is accepted in genetic toxicology that no single test system is capable of predicting with satisfactory reliability the genotoxic effects of a substance in humans. For this reason, test batteries are used when testing substances for possible genotoxic effects. The significance of the findings obtained can be clarified in three stages: interpretation of individual findings, assessment of genotoxicity data and evaluation with regard to the consequences on humans and the environment. Although these steps, which actually tend to merge into one another, cannot be strictly separated in practice, their differences must not be allowed to become too blurred and, in particu-
* The concept of this paper is based on the 'General strategy for the assessment of genotoxicity' (Mutation Res., 252 (1.991) 161-163). It was developed by the following members of the 'Commission for the Development of Guidelines for Genotoxicity Testing' of the German Society for Environmental Mutation Research (Gesellschafl fiir Umweltmutationsforschung, GUM). GUM Commission: P. Arni (Ciba-Geigy AG, Basel), R. Fahrig (chairman; Fraunhofer-Institut fiir Toxikologie und Aerosolforschung, Hannover), H.-R. Glatt (Universit~it Mainz), R. Lang (Schering AG, Berlin), S. Madle (BGA, Berlin), H.G. Miltenburger (TH Darmstadt), B.L. PoolZobel (Bundesforschungsanstalt fiir Ern~ihrung, Karlsruhe), F.E. Wiirgler (ETH und Universit~it Ziirich). Correspondence: Prof. 'Dr. F.E. Wiirgler, Institut fiir Toxikologie, ETH und Universit~it Ziirich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland.
lar, leaps from 'interpretation' to 'evaluation' should be avoided. Care should also be taken that assessment of data and evaluation of the consequences for man and environment are dependent on the test strategy chosen.
(1) Interpretation of individual findings Interpretations of individual genotoxicity findings are essentially descriptions of results which take into consideration the biological parameters which are significant for each test system used. This article is not concerned with interpretation criteria in particular, but we would like to summarize that the following points are also to be considered when interpreting. This is to ensure that the plausibility of a finding is thoroughly checked. - Is the result negative, equivocal or positive (weak or strong)? - Plausibility of method and result. - Type of d o s e - and time-effect relationship. - Are the sizes of random samples suitable for reliable detection of weak effects? - I n the case of in vitro findings: positive with/without external metabolizing system? - Reproducibility of an effect. - Dependence of an effect on concentrations which create drastically cytotoxic effects (in vitro) or doses which create acute systemic a n d / o r tissue-toxic effects (in vivo).
88 Statistical analyses regarding the probability of error can be of help when interpreting individual findings. However, the preconditions for selection of appropriate statistical methods are seldom granted. Vital importance should be attached to the biological relevance of the findings. (2) A s s e s s m e n t of genotoxicity data
(2.1) Fundamentals In order to arrive at a scientifically satisfactory assessment of whether or not a substance possesses genotoxic potential, it must be tested in various test systems which detect different genetic endpoints. The range and structure of the test battery suitable for the substance should allow for flexibility, where the following points, amongst others, should be taken into consideration: physico-chemical properties of the substance, structure comparison with known mutagens and non-mutagens, types and results of genotoxicity findings already obtained, other toxicological findings, the way the substance is used, the extent of the exposure to the substance and information/assessments concerning toxicokinetics (including foreign compound metabolism). The evaluation of genotoxic effects of substances is usually performed in two stages which, in practice, can merge into one another. (1) Basic testing for sensitive detection of genotoxic potential (hazard identification). As a rule, in vitro systems are employed which largely guarantee the availability of the substance (or its metabolites) for the test cells. Depending on the information gained from the above, further tests can become necessary for the following purposes. (2a) To investigate the postulated absence of a genotoxic effect. For this purpose, further tests are carried out while the biological parameters are varied: test cells (bacteria, mammalian cells in vitro and in vivo), metabolizing system ($9 mix, primary hepatocytes, intact animal metabolism), genetic endpoint (mutations, possibly also taking into account aneuploidy, indicator endpoints). (2b) To establish the significance of the positive results obtained. These tests should be purposely tailored to the type of positive finding as well as to the properties of the substance; here,
the biological conditions under which positive findings were attained have to be taken into consideration (test cells, genetic endpoint, metabolizing system).
(2.2) Basic testing for sensitive detection of genotoxic potential Basic testing of substances generally includes gene and chromosome mutation tests. Although most known mutagens induce both gene mutations and chromosomal aberrations, there is broad agreement in the scientific community that both endpoints must be analyzed at this stage and in separate systems. Certain substances are more likely to induce chromosomal aberrations while others tend toward induction of gene mutations, i.e., one of these two genetic endpoints is induced at lower concentrations than the other. A genotoxic potential could therefore be overlooked if only one of these endpoints were investigated.
(2.3) To investigate the postulated absence of genotoxic potential Should the basic testing prove negative, further tests may become necessary in order to check the assumption that no genotoxic potential is present. The decisive factor here is that the biological parameters vary. Basic testing is carried out predominantly with in vitro systems in which $9 mix (from Aroclor-induced rat liver) is used as an external metabolizing system. In tests which go beyond basic testing, other foreign compound metabolizing systems should also be incorporated; this is usually achieved by means of in vivo tests but is also possible using in vitro metabolizing systems, e.g., primary rat hepatocytes. In practice, the genetic endpoint often varies with the metabolizing system: -in order to take into account the in vivo metabolism, the bone marrow micronucleus test is most frequently used; - the main established rat hepatocyte test system is the UDS test which detects a particular type of DNA repair (excision repair). Since primary hepatocytes, however, are also suitable for cocultures with permanent cells, gene and chromosomal mutation tests with hepatocyte activation can also be carried out for specific problems.
89 W h e r e the endpoint gene mutation has been investigated with the basic testing in bacteria, a m a m m a l i a n cell test for gene mutations should be included in further tests. Besides chromosomal aberrations, micronucleus tests are also capable, in principle, of detecting aneuploidy. As long as no other aneuploidy tests on mammalian cells are available for routine performance, incorporation of an in vivo micronucleus test into test batteries is regarded as desirable so that aneugenic effects can at least be detected with the sensitivity characteristic of the micronucleus test. The following test battery often results from the performance of basic testing and further tests. gene mutation test in bacteria, gene mutation test in mammalian cell cultures, - chromosomal aberration test in vitro, - micronucleus test in vivo on the bone marrow. Besides tests which record mutations as genetic endpoints, indicator tests can also be used in further studies. In practice, great significance is attached above all to rat hepatocyte UDS tests in vitro and in v i v o / i n vitro, as these systems allow the liver metabolism to be taken into account. In vivo tests should only be carried out if it can be safely assumed that the test substance (or its metabolites) reaches the target cells in substantial amounts. As a matter of principle, further tests should be carried out in vitro where the available data regarding the physico-chemical and kinetic properties a n d / o r the findings from toxicological and pharmacological studies suggest a low systemic availability of the substance in vivo. If it is intended to consider the endpoint aneuploidy, this can also be done in in vitro micronucleus tests, the time schedule of which has been adapted to the detection of aneugen-induced micronuclei. -
-
(2.4) To establish the significance of positive findings The significance for man and the environment of positive genotoxicity findings must be clarified; with respect to the timepoint, type and extent of further tests, the aspects already mentioned are to be considered. For example, an increased aberration rate in a chromosomal aberration test in vitro, occurring only at the highest and cyto-
toxic concentration, is not nearly as suspect as a dose-dependent aberration rate increase. As a matter of principle, too much significance should not be attached to isolated positive in vitro findings. This is particularly true where a positive bacterial test conflicts with negative in vitro tests in mammalian cell cultures. While negative mammalian cell tests cannot simply 'override' the positive bacterial findings, our experience shows that such bacterial findings in many cases remain the only positive ones even after wide-ranging further testing (including in vivo tests). On the one hand, this may be explained by the fact that the bacterial test usually carried out - the A m e s test - was constructed in order to detect genotoxic effects with high sensitivity, thereby tending towards false positive findings. On the other hand, substances which induce neither gene nor chromosomal mutations in mammalian cell cultures are not generally expected to do so in vivo, either. Where tests are designed to establish the significance of positive genotoxicity findings, the genetic endpoint for which a positive effect was obtained, is also to be taken into account. Thus, the performance of a gene mutation test on mammalian cell cultures is r e c o m m e n d e d as a consequence of a positive bacterial gene mutation test. W h e r e clearly positive findings from chromosome aberration tests on mammalian cultures exist, a chromosome aberration test or micronucleus test in vivo should, as a rule, follow. It is usual practice to test in vitro mutagens in in vivo somatic cell tests. In our experience, however, the following aspects should be clarified before an in vivo test is carried out. (1) Reliability of the positive in vitro findings. Has it been established beyond all doubt that we are in fact dealing with an in vitro mutagen? If not, it is usually preferable to clarify the in vitro data. (2) Availability of the substance for the target cells. Can it be plausibly assumed that the substance or its metabolites reaches the target cells of the particular test system in any great amount (evaluation of absorption rate and, where appropriate, extent of the first-pass effect)? If not, no importance may be attached to a negative in vivo finding.
90 For substances with poor systemic availability, there are always problems in selecting tests suitable for the evaluation of positive in vitro findings. Appropriate measures can raise the systemic availability of many substances (e.g., selection of application route and formulation). However, the concentrations to be expected in the target ceils in vivo are often far below the concentrations which produced genotoxic effects in vitro. In these cases it has to be clarified whether or not the substance can have a genotoxic effect in the primarily exposed cells (e.g., in the gastrointestinal tract); this can be carried out with toxicokinetic considerations/investigations or, in individual cases, with genotoxicity tests. If an in vitro mutagen proves not to be systemically available, the risk to germ cells can be regarded as insignificant; in so far as somatic ceils are exposed to the substance, however, the suspicion of a carcinogenic risk continues to exist. If an in vitro mutagen is freely available systemically, it also possesses a basic potential to cause genotoxic effects in bone marrow, liver or embryonic cells. It must be assumed that such a substance can also exert genotoxic and carcinogenic effects in humans. As systemically freely available substances usually also reach the gonads and since it can be assumed that somatic cell mutagens also exert an effect on germ cells (provided they reach them), a risk of heritable germ cell mutations is to be expected. Genotoxicity tests on germ cells can become necessary in individual cases in order to check this expectation. Germ cell tests, however, usually only detect a particular spectrum of genetic damage and are almost exclusively carried out on male animals; for these reasons only limited significance is attached to individual negative findings.
(2.5) Avoidance of hasty evaluations A large number of varying test systems can be used in genotoxicity tests, in which the genetic endpoints, the test cells as well as the metabolizing systems can be varied. A decisive factor is that the individual findings obtained are only ever assessed in the light of the total data available. If no adequate test battery exists, the data are to be evaluated with care. This applies in the case of
negative findings (an adequate exclusion of a genotoxic effect at this stage is not even possible) as well as positive findings, the significance of which cannot yet be evaluated. In the past, much confusion was caused by frequent jumping to conclusions, where substances with individual (often even hardly plausible) positive findings were passed off as 'mutagens'. Especially if, for example, many genotoxicity findings are available for existing chemicals, data often seem to be contradictory at first sight. In our experience, the contradictions tend to resolve themselves if the individual findings are checked for their plausibility, unreliable findings are disregarded and if, in the case of contradictory in vitro and in vivo findings, substance availability for the relevant target cells is taken into consideration. (3) Evaluation with regard to the consequences for m a n and the environment
It is almost impossible to prove a justification for the extrapolation to humans of genotoxicity findings from model systems in vitro or in vivo. In particular, it has not yet been plausibly demonstrated that the exposure of humans to mutagens leads to inherited germ cell mutations. Although it is recognized that demonstration of such a causality in man is, for methodical reasons, almost impossible, it remains trying for the scientist that induced germ cell mutations, heritable in future generations, are only proven in animal models. Nor should the fact be overlooked that no quantifications of mutagenic/carcinogenic risks for humans can be undertaken with the routine methods of genetic toxicology. In order to understand the significance of genotoxic effects for carcinogenicity in humans, a series of examples has been gathered showing that mutations play a fundamental role in the multi-stage process of carcinogenesis. However, the basis for evaluation of positive genotoxicity findings with regard to carcinogenicity remains the fact that mutagens, as a rule, also lead to effects in the animal model long-term carcinogenicity study. The widely accepted social principle of prophylactic health protection has a strong influence
91 on the evaluation of positive genotoxicity findings. It is aimed to avoid as far as possible mutagenic/carcinogenic risks from chemicals in the environment; the desire to prevent exposure may lead to a ban. In the case of indispensable substances, risk management may become necessary. This can be carried out in a rather simple way if the genotoxic effect underlies a threshold mechanism; in any case, the plausible demonstration of a threshold is certainly very difficult in the individual case.
In practice, risk management often means risk minimization and definition of acceptable risks (or acceptable exposures). A rough estimation of the risks can be undertaken by comparing the lowest concentrations with observed genotoxic effects in the model systems with the highest expected concentrations to which human cells are exposed. If a very large difference is observed here, regarding the risk as acceptable may be justified, in carefully selected individual cases.
87
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1161/93/$06.00
MUTENV 08860
A s s e s s m e n t and evaluation of genotoxicity findings * Stephan Madle
a
and Rainer Lang b
a Federal Health Office, Max-uon-Pettenkofer-lnstitute, Berlin, Germany and b Schering AG, Experimental Toxicology, Berlin, Germany (Received 20 October 1992) (Accepted 21 October 1992)
Keywords: Interpretation of individual genotoxicity findings; Assessment of genotoxicity data; Evaluation of genotoxicity data
It is accepted in genetic toxicology that no single test system is capable of predicting with satisfactory reliability the genotoxic effects of a substance in humans. For this reason, test batteries are used when testing substances for possible genotoxic effects. The significance of the findings obtained can be clarified in three stages: interpretation of individual findings, assessment of genotoxicity data and evaluation with regard to the consequences on humans and the environment. Although these steps, which actually tend to merge into one another, cannot be strictly separated in practice, their differences must not be allowed to become too blurred and, in particu-
* The concept of this paper is based on the 'General strategy for the assessment of genotoxicity' (Mutation Res., 252 (1.991) 161-163). It was developed by the following members of the 'Commission for the Development of Guidelines for Genotoxicity Testing' of the German Society for Environmental Mutation Research (Gesellschafl fiir Umweltmutationsforschung, GUM). GUM Commission: P. Arni (Ciba-Geigy AG, Basel), R. Fahrig (chairman; Fraunhofer-Institut fiir Toxikologie und Aerosolforschung, Hannover), H.-R. Glatt (Universit~it Mainz), R. Lang (Schering AG, Berlin), S. Madle (BGA, Berlin), H.G. Miltenburger (TH Darmstadt), B.L. PoolZobel (Bundesforschungsanstalt fiir Ern~ihrung, Karlsruhe), F.E. Wiirgler (ETH und Universit~it Ziirich). Correspondence: Prof. 'Dr. F.E. Wiirgler, Institut fiir Toxikologie, ETH und Universit~it Ziirich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland.
lar, leaps from 'interpretation' to 'evaluation' should be avoided. Care should also be taken that assessment of data and evaluation of the consequences for man and environment are dependent on the test strategy chosen.
(1) Interpretation of individual findings Interpretations of individual genotoxicity findings are essentially descriptions of results which take into consideration the biological parameters which are significant for each test system used. This article is not concerned with interpretation criteria in particular, but we would like to summarize that the following points are also to be considered when interpreting. This is to ensure that the plausibility of a finding is thoroughly checked. - Is the result negative, equivocal or positive (weak or strong)? - Plausibility of method and result. - Type of d o s e - and time-effect relationship. - Are the sizes of random samples suitable for reliable detection of weak effects? - I n the case of in vitro findings: positive with/without external metabolizing system? - Reproducibility of an effect. - Dependence of an effect on concentrations which create drastically cytotoxic effects (in vitro) or doses which create acute systemic a n d / o r tissue-toxic effects (in vivo).
88 Statistical analyses regarding the probability of error can be of help when interpreting individual findings. However, the preconditions for selection of appropriate statistical methods are seldom granted. Vital importance should be attached to the biological relevance of the findings. (2) A s s e s s m e n t of genotoxicity data
(2.1) Fundamentals In order to arrive at a scientifically satisfactory assessment of whether or not a substance possesses genotoxic potential, it must be tested in various test systems which detect different genetic endpoints. The range and structure of the test battery suitable for the substance should allow for flexibility, where the following points, amongst others, should be taken into consideration: physico-chemical properties of the substance, structure comparison with known mutagens and non-mutagens, types and results of genotoxicity findings already obtained, other toxicological findings, the way the substance is used, the extent of the exposure to the substance and information/assessments concerning toxicokinetics (including foreign compound metabolism). The evaluation of genotoxic effects of substances is usually performed in two stages which, in practice, can merge into one another. (1) Basic testing for sensitive detection of genotoxic potential (hazard identification). As a rule, in vitro systems are employed which largely guarantee the availability of the substance (or its metabolites) for the test cells. Depending on the information gained from the above, further tests can become necessary for the following purposes. (2a) To investigate the postulated absence of a genotoxic effect. For this purpose, further tests are carried out while the biological parameters are varied: test cells (bacteria, mammalian cells in vitro and in vivo), metabolizing system ($9 mix, primary hepatocytes, intact animal metabolism), genetic endpoint (mutations, possibly also taking into account aneuploidy, indicator endpoints). (2b) To establish the significance of the positive results obtained. These tests should be purposely tailored to the type of positive finding as well as to the properties of the substance; here,
the biological conditions under which positive findings were attained have to be taken into consideration (test cells, genetic endpoint, metabolizing system).
(2.2) Basic testing for sensitive detection of genotoxic potential Basic testing of substances generally includes gene and chromosome mutation tests. Although most known mutagens induce both gene mutations and chromosomal aberrations, there is broad agreement in the scientific community that both endpoints must be analyzed at this stage and in separate systems. Certain substances are more likely to induce chromosomal aberrations while others tend toward induction of gene mutations, i.e., one of these two genetic endpoints is induced at lower concentrations than the other. A genotoxic potential could therefore be overlooked if only one of these endpoints were investigated.
(2.3) To investigate the postulated absence of genotoxic potential Should the basic testing prove negative, further tests may become necessary in order to check the assumption that no genotoxic potential is present. The decisive factor here is that the biological parameters vary. Basic testing is carried out predominantly with in vitro systems in which $9 mix (from Aroclor-induced rat liver) is used as an external metabolizing system. In tests which go beyond basic testing, other foreign compound metabolizing systems should also be incorporated; this is usually achieved by means of in vivo tests but is also possible using in vitro metabolizing systems, e.g., primary rat hepatocytes. In practice, the genetic endpoint often varies with the metabolizing system: -in order to take into account the in vivo metabolism, the bone marrow micronucleus test is most frequently used; - the main established rat hepatocyte test system is the UDS test which detects a particular type of DNA repair (excision repair). Since primary hepatocytes, however, are also suitable for cocultures with permanent cells, gene and chromosomal mutation tests with hepatocyte activation can also be carried out for specific problems.
89 W h e r e the endpoint gene mutation has been investigated with the basic testing in bacteria, a m a m m a l i a n cell test for gene mutations should be included in further tests. Besides chromosomal aberrations, micronucleus tests are also capable, in principle, of detecting aneuploidy. As long as no other aneuploidy tests on mammalian cells are available for routine performance, incorporation of an in vivo micronucleus test into test batteries is regarded as desirable so that aneugenic effects can at least be detected with the sensitivity characteristic of the micronucleus test. The following test battery often results from the performance of basic testing and further tests. gene mutation test in bacteria, gene mutation test in mammalian cell cultures, - chromosomal aberration test in vitro, - micronucleus test in vivo on the bone marrow. Besides tests which record mutations as genetic endpoints, indicator tests can also be used in further studies. In practice, great significance is attached above all to rat hepatocyte UDS tests in vitro and in v i v o / i n vitro, as these systems allow the liver metabolism to be taken into account. In vivo tests should only be carried out if it can be safely assumed that the test substance (or its metabolites) reaches the target cells in substantial amounts. As a matter of principle, further tests should be carried out in vitro where the available data regarding the physico-chemical and kinetic properties a n d / o r the findings from toxicological and pharmacological studies suggest a low systemic availability of the substance in vivo. If it is intended to consider the endpoint aneuploidy, this can also be done in in vitro micronucleus tests, the time schedule of which has been adapted to the detection of aneugen-induced micronuclei. -
-
(2.4) To establish the significance of positive findings The significance for man and the environment of positive genotoxicity findings must be clarified; with respect to the timepoint, type and extent of further tests, the aspects already mentioned are to be considered. For example, an increased aberration rate in a chromosomal aberration test in vitro, occurring only at the highest and cyto-
toxic concentration, is not nearly as suspect as a dose-dependent aberration rate increase. As a matter of principle, too much significance should not be attached to isolated positive in vitro findings. This is particularly true where a positive bacterial test conflicts with negative in vitro tests in mammalian cell cultures. While negative mammalian cell tests cannot simply 'override' the positive bacterial findings, our experience shows that such bacterial findings in many cases remain the only positive ones even after wide-ranging further testing (including in vivo tests). On the one hand, this may be explained by the fact that the bacterial test usually carried out - the A m e s test - was constructed in order to detect genotoxic effects with high sensitivity, thereby tending towards false positive findings. On the other hand, substances which induce neither gene nor chromosomal mutations in mammalian cell cultures are not generally expected to do so in vivo, either. Where tests are designed to establish the significance of positive genotoxicity findings, the genetic endpoint for which a positive effect was obtained, is also to be taken into account. Thus, the performance of a gene mutation test on mammalian cell cultures is r e c o m m e n d e d as a consequence of a positive bacterial gene mutation test. W h e r e clearly positive findings from chromosome aberration tests on mammalian cultures exist, a chromosome aberration test or micronucleus test in vivo should, as a rule, follow. It is usual practice to test in vitro mutagens in in vivo somatic cell tests. In our experience, however, the following aspects should be clarified before an in vivo test is carried out. (1) Reliability of the positive in vitro findings. Has it been established beyond all doubt that we are in fact dealing with an in vitro mutagen? If not, it is usually preferable to clarify the in vitro data. (2) Availability of the substance for the target cells. Can it be plausibly assumed that the substance or its metabolites reaches the target cells of the particular test system in any great amount (evaluation of absorption rate and, where appropriate, extent of the first-pass effect)? If not, no importance may be attached to a negative in vivo finding.
90 For substances with poor systemic availability, there are always problems in selecting tests suitable for the evaluation of positive in vitro findings. Appropriate measures can raise the systemic availability of many substances (e.g., selection of application route and formulation). However, the concentrations to be expected in the target ceils in vivo are often far below the concentrations which produced genotoxic effects in vitro. In these cases it has to be clarified whether or not the substance can have a genotoxic effect in the primarily exposed cells (e.g., in the gastrointestinal tract); this can be carried out with toxicokinetic considerations/investigations or, in individual cases, with genotoxicity tests. If an in vitro mutagen proves not to be systemically available, the risk to germ cells can be regarded as insignificant; in so far as somatic ceils are exposed to the substance, however, the suspicion of a carcinogenic risk continues to exist. If an in vitro mutagen is freely available systemically, it also possesses a basic potential to cause genotoxic effects in bone marrow, liver or embryonic cells. It must be assumed that such a substance can also exert genotoxic and carcinogenic effects in humans. As systemically freely available substances usually also reach the gonads and since it can be assumed that somatic cell mutagens also exert an effect on germ cells (provided they reach them), a risk of heritable germ cell mutations is to be expected. Genotoxicity tests on germ cells can become necessary in individual cases in order to check this expectation. Germ cell tests, however, usually only detect a particular spectrum of genetic damage and are almost exclusively carried out on male animals; for these reasons only limited significance is attached to individual negative findings.
(2.5) Avoidance of hasty evaluations A large number of varying test systems can be used in genotoxicity tests, in which the genetic endpoints, the test cells as well as the metabolizing systems can be varied. A decisive factor is that the individual findings obtained are only ever assessed in the light of the total data available. If no adequate test battery exists, the data are to be evaluated with care. This applies in the case of
negative findings (an adequate exclusion of a genotoxic effect at this stage is not even possible) as well as positive findings, the significance of which cannot yet be evaluated. In the past, much confusion was caused by frequent jumping to conclusions, where substances with individual (often even hardly plausible) positive findings were passed off as 'mutagens'. Especially if, for example, many genotoxicity findings are available for existing chemicals, data often seem to be contradictory at first sight. In our experience, the contradictions tend to resolve themselves if the individual findings are checked for their plausibility, unreliable findings are disregarded and if, in the case of contradictory in vitro and in vivo findings, substance availability for the relevant target cells is taken into consideration. (3) Evaluation with regard to the consequences for m a n and the environment
It is almost impossible to prove a justification for the extrapolation to humans of genotoxicity findings from model systems in vitro or in vivo. In particular, it has not yet been plausibly demonstrated that the exposure of humans to mutagens leads to inherited germ cell mutations. Although it is recognized that demonstration of such a causality in man is, for methodical reasons, almost impossible, it remains trying for the scientist that induced germ cell mutations, heritable in future generations, are only proven in animal models. Nor should the fact be overlooked that no quantifications of mutagenic/carcinogenic risks for humans can be undertaken with the routine methods of genetic toxicology. In order to understand the significance of genotoxic effects for carcinogenicity in humans, a series of examples has been gathered showing that mutations play a fundamental role in the multi-stage process of carcinogenesis. However, the basis for evaluation of positive genotoxicity findings with regard to carcinogenicity remains the fact that mutagens, as a rule, also lead to effects in the animal model long-term carcinogenicity study. The widely accepted social principle of prophylactic health protection has a strong influence
91 on the evaluation of positive genotoxicity findings. It is aimed to avoid as far as possible mutagenic/carcinogenic risks from chemicals in the environment; the desire to prevent exposure may lead to a ban. In the case of indispensable substances, risk management may become necessary. This can be carried out in a rather simple way if the genotoxic effect underlies a threshold mechanism; in any case, the plausible demonstration of a threshold is certainly very difficult in the individual case.
In practice, risk management often means risk minimization and definition of acceptable risks (or acceptable exposures). A rough estimation of the risks can be undertaken by comparing the lowest concentrations with observed genotoxic effects in the model systems with the highest expected concentrations to which human cells are exposed. If a very large difference is observed here, regarding the risk as acceptable may be justified, in carefully selected individual cases.