ToxSci Advance Access originally published online on February 14, 2007
Toxicological Sciences 2007 97(2):237-240; doi:10.1093/toxsci/kfm019
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Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part IV:Recommendation of a Working Group of the Gesellschaft fuer Umwelt-Mutationsforschung (GUM) for a Simple and Straightforward Approach to Genotoxicity Testing
* P&G Prestige and Professional, Cosmital SA, Experimental Product Safety, CH-1723 Marly, Switzerland
Department of Toxicology, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland
Kao Professional Salon Services GmbH, D-64297 Darmstadt, Germany
Bayer Healthcare AG, D-42096 Wuppertal, Germany
¶ Federal Institute for Risk Assessment (BfR), Safety of Substances and Preparations, D-14195 Berlin, Germany
|| Institute of Toxicology, Merck KGaA, D-64271 Darmstadt, Germany
||| RCC Cytotest Cell Research GmbH, D-64380 Rossdorf, Germany
1 To whom correspondence should be addressed at Department of RD-EPS, P&G Prestige and Professional, Cosmital SA, Rte de Chesalles 21, CH-1723 Marly, Switzerland. Fax: +41 26 435 26 66. E-mail: pfuhler.s{at}pg.com.
Received December 21, 2006; accepted February 12, 2007
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ABSTRACT |
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Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.
Key Words: genotoxicity testing; testing strategies; hazard identification.
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INTRODUCTION |
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Identification of possible genotoxic effects has long been fundamental for toxicity testing of chemical substances. Originally, genotoxicity testing focused on the detection of germ cell mutagens. In current regulatory practice, emphasis is laid on screening for possible carcinogenic substances. Furthermore, genotoxicity testing is increasingly being used to clarify the significance of equivocal findings in carcinogenicity studies or whether carcinogenic effects are linked to genotoxicity. In recent years, genotoxic effects are also discussed as a cause for other diseases (Erickson, 2003
).
Genotoxicity includes induction of mutations (gene mutations, structural chromosome mutations, and genome mutations) as well as so-called indicator effects which are related to mutations (e.g., induction of DNA modifications, DNA repair, and recombination). Various genotoxicity testing strategies have been published (e.g., COM, 2000; European Commission, 2003; Fahrig et al., 1991; ICH guideline S2B, 1997; Kirkland et al., 2005b). A common approach is to subdivide genotoxicity tests in "basic testing" and "follow-up testing." In basic testing mutation endpoints are usually investigated while indicator tests (besides mutagenicity tests) may be used in follow-up testing (e.g., for clarification of the significance of positive findings). Indicator tests comprise a broader methodological spectrum and enable the detection of genotoxic effects in nonproliferating cells and in a wider variety of organs and cell types. Therefore, such indicator tests are useful tools for the investigation of local genotoxic effects that might be directly related to organ-specific carcinogenic effects.
For well-established and long-standing genetic toxicity tests, guidelines of the Organisation for Economic Cooperation and Development (OECD) are available which describe worldwide-accepted methodological standards. For some other well-established tests, OECD guidelines are in preparation (e.g., for the in vitro micronucleus test [MNT]) or recommendations by international expert groups are available (e.g., for the comet assay: Hartmann et al., 2003; Tice et al., 2000). At present, different genotoxicity-testing strategies are applied depending on the regulated "substance class" (use of substances, type and degree of exposure, risk-benefit considerations, etc.). For all the different testing strategies, at least two genotoxicity tests are needed for basic testing in order to cover both gene and chromosome mutations. In some cases, e.g., for pharmaceuticals and veterinary drugs, an in vivo test is considered mandatory in basic testing (Müller et al., 1999). For several substance classes, e.g., pesticides, biocides, and food contact materials, a second in vitro mammalian cell test is required. Recent publications demonstrate that the in vitro mammalian cell tests, while showing high sensitivity, tend toward creating irrelevant positive results (Kirkland et al., 2005a; Matthews et al., 2006). The combination of two in vitro mammalian assays leads to 75% or more positive results for noncarcinogens, which leads to unnecessary follow-up in animal tests. This is an argument against such an approach.
In 1991, the German-speaking section of the European Environmental Mutagen Society has published a general strategy for the assessment of genotoxicity (Fahrig et al., 1991). Based on regulatory demands for the various substance classes and the scientific state of the art, harmonization was recommended. Emphasis was laid on flexibility (in order to cover different regulatory demands and preferences of test laboratories) and the necessity of a weight of evidence approach—to evaluate single findings in the context of the whole data set.
In the meantime, comprehensive experience has been gained with genotoxicity testing for regulatory purposes and new test methods are available for routine testing. Two new methods with specific advantages are the in vitro MNT and the in vivo comet assay. The in vivo comet assay enables the investigation of local genotoxic effects in almost any tissue (Brendler-Schwaab et al., 2005). The in vitro MNT is advantageous because it detects both clastogenic and aneugenic effects (Kirsch-Volders et al., 2003) and is a sensitive tool to detect carcinogens (Kirkland et al., 2005a; Matthews et al., 2006). Based on these methods and regulatory experience among the authors, the present paper suggests a simple and straightforward testing strategy which can be broadly applied.
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TESTING APPROACH |
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Genotoxicity testing usually comprises two phases, basic testing (stage I) and follow-up testing (stage II). A combination of genotoxicity tests is necessary to cover all types of mutations (i.e., gene mutations, structural chromosome aberrations, and genome mutations). Rigorous protocols should be used in order to optimize the capability for detecting mutagenic potential and to ensure that a negative result can be accepted with confidence. The recommended strategy is outlined in Figure 1.
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Stage I (Basic Testing)
Stage I testing is performed to detect the potential of a compound to cause mutations and will usually comprise a combination of in vitro tests. In recent years, it was emphasized that not only gene mutations and structural chromosome aberrations but also numeric chromosome aberrations (aneuploidy) should be covered in initial testing. The in vitro MNT is able to detect clastogens and aneugens. Its introduction into routine genotoxicity testing as an alternative for the in vitro chromosome aberration test has therefore been suggested (COM, 2000; European Commission, 2003
). This method has recently been validated by the European Center for Validation of Alternative Methods (ESAC, 2007). An OECD guideline is currently in preparation. The most efficient way to consider all types of mutations is to combine a bacterial gene mutation test with an in vitro MNT in mammalian cells. The main difference to some testing strategies is the lack of a mammalian cell gene mutation test. A mammalian cell gene mutation test appears to be dispensable in stage I testing because the bacterial gene mutation test detects all relevant modes of action specifically leading to gene mutations. Moreover, most of the substances positive in mammalian gene mutation tests also induce clastogenic effects and these compounds will be detected with a high degree of sensitivity by the in vitro MNT.
An appropriate metabolic activation of the test compound has to be ensured. The standard S9-mix is usually sufficient for the detection of indirect mutagens. For specific compounds which may not be adequately metabolized by standard S9-mix, appropriate modifications of the metabolic activation system should be considered. Modifications may include the use of variations in S9-mix, freshly isolated hepatocytes or cell lines with specific enzymatic capacities.
In general, stage I testing will lead to results which can be interpreted unequivocally as negative or positive. Equivocal results should be clarified by repeating the respective test (possibly considering modifications of the test protocol). If despite these efforts no clear positive or negative test result can be achieved, an additional in vitro test with the genetic end point of concern—or alternatively an in vivo assay—should be performed.
If the results of the stage I testing do not provide any hint of a mutagenic potential of the test compound, there is no need for any additional confirmatory (stage II) testing. Points to consider at this stage are whether there are relevant limitations in the reliability of the test result and whether there are any specific factors that would make a nonroutine testing strategy necessary (e.g., substance class–specific factors, inappropriate representation of in vivo metabolism).
Positive findings in stage I tests will, in general, need further evaluation in order to clarify their relevance for the in vivo situation. This includes further assessment of the results and/or additional testing (stage II).
Stage II (Follow-Up Testing)
The choice of the appropriate assays for stage II will depend on the outcome of the stage I testing and considerations of the toxicokinetic properties of the compound. In the following paragraphs, follow-up strategies (stage II testing) are described for the different scenarios where positive findings were demonstrated in stage I.
Scenario 1: Negative bacterial gene mutation assay, negative in vitro MNT.
In general no further testing is necessary.
Scenario 2: Positive bacterial gene mutation assay, negative in vitro MNT.
In this situation, performance of a mammalian cell gene mutation assay is suggested to clarify whether the result obtained in bacteria is relevant for mammalian cells. Both the hypoxanthine-guanine phosphoribosyl transferase test and the L5178Y TK+/– test (mouse lymphoma assay) are considered equally adequate for this purpose. If the mammalian cell gene mutation test is negative (in addition to the negative in vitro MNT), a mutagenic potential of the test compound in mammalian cells can be excluded unless there are other compound-specific reasons for concern. If the mammalian cell gene mutation assay is positive, further testing will usually be necessary (see Scenario 3).
Scenario 3: Positive in vitro MNT, negative or positive bacterial gene mutation assay.
Stage II testing will usually be required to follow up on the positive findings unless it can be shown that the positive result from in vitro is not relevant for in vivo. Before the decision to carry out an in vivo study is made, it is necessary to carefully consider the degree of systemic availability of the test substance, possible metabolic pathways, the specific genetic end point, and which organs will be exposed.
In order to obtain the maximum information from a single in vivo study, a combination of the in vivo MNT and in vivo comet assay is proposed. Such a combination covers systemic genotoxic effects and local effects (site of contact tissue and target organ for toxicity) as well as different genetic mechanisms. The DNA strand breaks measured by the comet assay are primary DNA lesions with relevance for the formation of gene and chromosome mutations, and micronuclei indicate clastogenic and/or aneugenic events.
The test performance, including evaluation and interpretation of the results, should be in accordance with the guideline for the in vivo MNT and should meet the requirements for an appropriate use of the in vivo comet assay (Hartmann et al., 2003; Tice et al., 2000). A possible test design is to administer the test substance three times to each animal 48, 24, and 3–6 h prior to sacrifice. However, specific toxicokinetic or toxicological properties of a compound may favor single application or separate studies for the two end points.
Micronuclei will be measured in bone marrow while the comet assay will usually be performed with cells from a directly exposed tissue (in general the gastrointestinal tract or the respiratory tract) and the liver as a reference organ. If known, the potential target organ for carcinogenesis should also be evaluated. Current developments enable micronucleus determination in tissues other than the bone marrow (e.g., liver, colon, and skin) and may be applied as a useful addition to the standard approach. Transgenic mutation assays (recent review: Lambert et al., 2005) may be considered as an alternative to the in vivo comet assay. However, the comet assay's ease of use and the possibility to combine it with the MNT (i.e., to save animals, cost and time) as well as its ability to detect a broader spectrum of primary DNA alterations are arguments in its favor.
Negative results for such a combined study in relevant tissues (target cells should be exposed) will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Positive results have to be evaluated case by case considering, among other things, the positive genetic end point, local versus systemic effects, and the type of positive in vitro data. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.
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ACKNOWLEDGMENTS |
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The authors like to thank Guenter Speit and Peter Kasper for their valuable contributions and the Gesellschaft fuer Umwelt-Mutationsforschung for supporting this activity.
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REFERENCES |
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