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Mutagenesis Advance Access originally published online on June 17, 2009
Mutagenesis 2009 24(4):341-349; doi:10.1093/mutage/gep014
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© World Health Organization, 2009. All rights reserved. The World Health Organization has granted the publisher permission for the reproduction of this article.

Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme

David A. Eastmond, Andrea Hartwig1, Diana Anderson2, Wagida A. Anwar3, Michael C. Cimino4, Ivan Dobrev5, George R. Douglas6, Takehiko Nohmi7, David H. Phillips8 and Carolyn Vickers9,*

Environmental Toxicology Graduate Program, University of California, Riverside, CA, USA 1Institut für Lebensmitteltechnologie und Lebensmittelchemie, Technische Universität Berlin, Berlin, Germany 2Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, UK 3Department of Community, Environmental and Occupational Medicine, Faculty of Medicine, Ain Shams University, Abassya, Cairo, Egypt 4Risk Assessment Division, Science Support Branch, Office of Pollution Prevention and Toxics, Environmental Protection Agency, Washington, DC, USA 5Department of Chemical Risk Assessment, Fraunhofer Institute for Toxicology and Experimental Medicine, Hanover, Germany 6Mechanistic Studies Division, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario, Canada 7Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan 8Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, UK 9International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.


   Introduction
Top
 Introduction
Strategy for mutagenicity...
 Funding
 References
 
Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing (1Go), there have been a number of publications addressing test strategies for mutagenicity (2Go–6Go) and reviews thereof (7Go). In addition, analyses of test batteries and their correlation with carcinogenicity (8Go–11Go) have indicated that an optimal solution to this issue has not yet been found. The 2005 International Workshop on Genotoxicity Testing (IWGT) meeting in San Francisco, USA, discussed many of these problems, and reports of this meeting (10Go,12Go) and companion papers (13Go–16Go) have recently been published.

Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation (i.e. point mutations or deletions/insertions that affect single or blocks of genes), clastogenicity (i.e. structural chromosome changes) and aneuploidy (i.e. numerical chromosome aberrations). It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. This is not surprising, as a wide variety of possible genetic events can occur. For example, some mutagens preferentially induce gene mutations by either base pair substitutions or frameshift mechanisms, whereas others induce chromosome mutations but show little or no evidence of inducing gene mutations.

The World Health Organization (WHO) therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing (1Go) as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. A public review draft paper was prepared by an International Drafting Group Meeting of experts, held at the Fraunhofer Institute for Toxicology and Experimental Medicine in Hanover, Germany, on April 11–12, 2007, and revised, following peer and public review, by an expert review meeting hosted by the University of Bradford, Bradford, UK, on June 30 to July 1, 2008. The present paper is the product of the expert review meeting.


   Strategy for mutagenicity testing
Top
 Introduction
 Strategy for mutagenicity...
Funding
 References
 
The approach presented in this paper (see Figure 1) focuses on the identification of mutagens and genotoxic carcinogens. The term ‘mutation’ as understood in this paper (a glossary of terms used in this paper is available on the IPCS website at http://www.who.int/ipcs/publications/methods/harmonization/en/index.html) refers to permanent changes in the structure and/or amount of the genetic material of an organism that can lead to heritable changes in its function, and it includes gene mutations as well as structural and numerical chromosome alterations. The group is aware of other mechanisms leading to carcinogenicity and other heritable diseases, but their identification requires additional types of mechanistic studies. ‘Genotoxicity’ refers to the capability of substances to damage DNA and/or cellular components regulating the fidelity of the genome—such as the spindle apparatus, topoisomerases, DNA repair systems and DNA polymerases (4Go)—and includes all adverse effects on genetic information. These potentially harmful effects on genetic material may be mediated directly or indirectly and are not necessarily associated with mutagenicity. Genotoxicity is therefore a broader term than ‘mutagenicity’, which refers to the capacity to give rise to mutations.


Figure 1
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  		Fig. 1. Strategy for mutagenicity testing.

 
Because of the wide range of genetic damage that can occur, test batteries are designed to include complementary tests evaluating different mechanisms of mutagenicity. At all stages of the outlined testing strategy, a weight of evidence approach and scientific judgement should be used. Multiple negative results may not be sufficient to remove concern for mutagenicity raised by a clear positive result in a single mutagenicity assay.

Most short-term tests in bacteria and mammalian cell cultures have been designed primarily for hazard identification and thus can represent only the starting point in the process of risk assessment. Whether or not the observed effects are relevant for humans under anticipated exposure conditions depends on pharmacokinetic, pharmacodynamic and other factors that require investigation in vivo.

Especially when choosing in vivo assays and when proceeding into germ cell mutagenicity studies (see Strategy for germ cell testing), expert judgement is required to select the appropriate test systems and to avoid uninformative and thus unnecessary animal experiments.

Development of a testing strategy
Before initiating mutagenicity testing on a particular substance (or mixture of substances), the following aspects should be considered, when available:

(i) Chemical structure and class of the substance (possible structure–activity relationships) and physicochemical properties, such as solubility and stability;
(ii) Expected pathways of metabolism, chemical and biological reactivity/activity and relationship to known genotoxic substances and
(iii) Routes of exposure, bioavailability and target tissues for genotoxicity.

Critical evaluation of available data prior to testing usually provides important information for choosing the appropriate in vitro assays, but even more so for the selection of appropriate in vivo studies.

Distinction needs to be made between ‘mutagenicity tests’ in the strict sense and ‘indicator tests’ that provide evidence of interaction with DNA that may or may not lead to mutations (e.g. DNA adducts, DNA strand breaks and sister chromatid exchanges). Preference should be given to mutagenicity tests whenever possible.

In vitro testing
Usually two or three different tests in bacteria and mammalian cells are selected to cover the end points of gene mutations, clastogenicity (structural chromosome aberrations) and aneuploidy (numerical chromosome aberrations), taking into account physicochemical properties of substances under consideration.

In vitro tests. Screening should be based on a limited number of tests that are well validated and informative. Genotoxicity test batteries generally include the following:

(i) A test for gene mutation in bacteria (bacterial reverse mutation assay): Organisation for Economic Co-operation and Development (OECD) Test Guideline 471 recommends the use of at least five strains of bacteria: (a) Salmonella typhimurium TA1535, (b) S.typhimurium TA1537 or TA97 or TA97a, (c) S.typhimurium TA98, (d) S.typhimurium TA100 and (e) Escherichia coli WP2 or E.coli WP2uvrA or S.typhimurium TA102. The choice of additional tests depends on the chemical structure and class of the substance (see Development of a testing strategy). Table I describes the most commonly used bacterial mutagenicity tests.
(ii) In vitro mammalian assays: These assays should evaluate the potential of a substance to induce point mutations, clastogenicity and/or aneugenicity, by using either mammalian cell lines or primary human cell cultures such as fibroblasts or lymphocytes (e.g. mouse lymphoma thymidine kinase assay, hypoxanthine guanine phosphoribosyltransferase assay or cytogenetic evaluation of chromosomal damage in mammalian cells via either the in vitro chromosome aberration or the in vitro micronucleus test) (see Table II).


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  		Table I. Common in vitro bacterial assays

 


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  		Table II. Common in vitro mammalian assays

 
Evaluation of in vitro testing results. In the evaluation, results are classified into (i) positive, (ii) negative and (iii) contradictory or equivocal:
(i) Positive: Substance is positive at one or more end points of mutagenicity.
(ii) Negative: Substance is negative in all test systems under appropriate in vitro test conditions; the substance is not mutagenic (or genotoxic) in vitro and is anticipated not to be mutagenic in vivo [for exceptions, see refs (37Go,38Go)].
(iii) Contradictory or equivocal (e.g. borderline biological or statistical significance): All other substances.

Follow-up to in vitro testing.

(i) Positive in vitro results
In vivo test; selection of an appropriate end point; if necessary, further in vitro studies to optimize in vivo testing (e.g. kinetochore staining as an addition in the micronucleus assay of in vitro aneugens). Follow-up tests in vitro may also provide additional mechanistic information to enable interpretation of a positive finding.
(ii) Negative in vitro results
In vivo testing is recommended in the case of ‘high’ or ‘moderate and sustained’ human exposure or for substances otherwise of high concern. In limited cases, metabolic considerations may trigger in vivo testing (38Go).
(iii) Contradictory or equivocal in vitro results
Further in vitro testing to clarify positive or negative results; depending on whether the situation is resolved by further in vitro testing, proceed according to ‘positive’ or ‘negative’.

In vivo testing
In vivo tests. In vivo tests (see Tables III and IV) should be chosen carefully to avoid an uninformative outcome and with concern for animal welfare. Therefore, toxicokinetics, metabolism and chemical reactivity have to be considered carefully. In vivo tests may also be used for evaluation of a dose–response, species differences or mode of action determination. The use of such tests needs to be considered on a case-by-case basis for risk assessment purposes.


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  		Table III. Common in vivo genotoxicity assays

 


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  		Table IV. Germ cell assays

 
The choice of an in vivo follow-up test should be guided by the spectrum of genotoxic events observed in the in vitro studies as well as knowledge of the bioavailability, distribution, metabolism and target organ specificity of the substance. Typically, a bone marrow micronucleus or clastogenicity test is conducted. However, if there are indications that point to a more appropriate assay, then this assay should be conducted instead (e.g. mutagenicity study with transgenic animals and/or comet assay in potential target tissues).

Follow-up to in vivo testing.

(i) Positive in vivo results
Substance is considered an ‘in vivo somatic cell mutagen’. Testing for germ cell mutagenicity (see Strategy for germ cell testing) may be required.
(ii) Negative in vivo results
Further in vivo testing is recommended in the case of positive in vitro studies. Again, the second in vivo test is chosen on a case-by-case basis, as stated above. If the test is negative, it is concluded that there is no evidence for in vivo mutagenicity.
(iii) Equivocal in vivo results
Equivocal results may be due to low statistical power, which can be improved by increasing the number of treated animals and/or scored cells.
If the situation is unresolved, a second in vivo test is recommended, chosen on a case-by-case basis (ordinarily on a different end point or in a different tissue, depending on toxicokinetics, metabolism and mode of action); proceed according to ‘positive’ or ‘negative’.

Strategy for germ cell testing
When information on the risk to the offspring of exposed individuals is important, the following germ cell testing strategy is recommended.

For substances that give positive results for mutagenic effects in somatic cells in vivo, their potential to affect germ cells should be considered. If there is toxicokinetic or toxicodynamic evidence that germ cells are actually exposed to the somatic mutagen or its bioactive metabolites, it is reasonable to assume that the substance may also pose a mutagenic hazard to germ cells and thus a risk to future generations.

Where germ cell testing is indicated, judgement should be used to select the most appropriate test strategy. There are a number of tests available (summarized in Table IV), which fall into two classes:

(i) Tests in germ cells per se (class 1)
(ii) Tests to detect effects in the offspring (or potential offspring) of exposed animals (class 2)

Three tests that are available for such studies have established OECD test guidelines:

(i) Clastogenicity in rodent spermatogonial cells (class 1): OECD Test Guideline 483 (65Go)
(ii) The dominant lethal test (class 2): OECD Test Guideline 478 (66Go)
(iii) The mouse heritable translocation assay (class 2): OECD Test Guideline 485 (67Go)

The above-mentioned class 2 tests usually require large numbers of animals. Thus, in order to minimize the use of animals in germ cell testing, it is advisable to start with tests that detect effects in germ cells per se (class 1). Other methods include (but are not limited to) gene mutation tests in transgenic animals [see ref. (41Go) for IWGT guidance], gene mutations in the more recent Expanded Simple Tandem Repeat (ESTR) assay, chromosomal assays (including those using fluorescence in situ hybridization), comet assay and DNA adduct analysis.

Following the use of such tests, if quantification of heritable effects is required (class 2), an assay for ESTR mutations can be performed with the offspring of a low number of exposed animals. Tests used historically to investigate transmitted effects (e.g. the heritable translocation test and the specific locus test) can also be performed; however, they use large numbers of animals.

Class 1 and class 2 germ cell assays are summarized in Table IV. The strategy used in germ cell mutagenicity testing is outlined in Figure 2.


Figure 2
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  		Fig. 2. Strategy in germ cell mutagenicity testing.

 

   Funding
Top
 Introduction
 Strategy for mutagenicity...
 Funding
References
 
This work was funded by donations to the World Health Organization by a number of member states of the World Health Assembly.


   Acknowledgments
 
This publication contains the collective views of the listed authors and does not necessarily represent the decisions or stated policies of the World Health Organization or of the authors' affiliated agencies or institutions.

WHO thanks the Fraunhofer Institute for Toxicology and Experimental Medicine, Hanover, Germany, for its assistance in preparing for and hosting the international drafting group expert meeting that developed the first draft of this paper. Participants in the drafting group meeting were M.C.C., Environmental Protection Agency, Washington, DC, USA; G.R.D., Health Canada, Ottawa, Ontario; D.A.E., University of California, Riverside, CA, USA; A.H., Technische Universität Berlin, Berlin; Janet Kielhorn, Fraunhofer Institute for Toxicology and Experimental Medicine, Hanover; Andreas Luch, Federal Institute for Risk Assessment, Berlin; D.H.P., Institute of Cancer Research, Sutton; Atsuya Takagi, National Institute of Health Sciences, Tokyo; Raymond Tennant, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, USA; and C.V., International Programme on Chemical Safety, WHO, Geneva. WHO also thanks the University of Bradford, Bradford, UK, for hosting the expert meeting that finalized this paper. Participants at that meeting are listed as the authors of the paper. Laurence Musset of the OECD was an observer at that meeting and provided information relevant to the OECD Test Guidelines. Professor David Kirkland was invited to the drafting group meeting and the expert review meeting in his capacity as Steering Committee Chair for the International Workshops on Genotoxicity Testing. He did not participate in the decision-making parts of the meetings. Finally, WHO expresses its appreciation to D.A.E. for chairing and to A.H. for acting as rapporteur for both meetings.

Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +41 22 791 1286; Fax: +41 22 791 4848; Email: ipcsmail{at}who.int


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 Strategy for mutagenicity...
 Funding
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Received on February 2, 2009; revised on April 14, 2009; accepted on April 15, 2009.


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