Germ cell mutagens/genotoxins are substances that cause heritable (passed on to progeny) changes in the genetic material in the germ cells, namely spermatocytes or oocytes (UNECE, 2004; ECVAM, 2002). The term mutagen is used to describe a substance that induces transmissible changes in the structure of the DNA (Maurici et al., 2005b) that may involve a single gene or a group of genes. Genotoxins are a broader category of substances that induce changes to the structure or number of genes via chemical interaction with DNA and/or non-DNA targets (Maurici et al., 2005b). The term "genotoxicity" is generally used unless a specific assay for mutations is being discussed.

At the early stages of testing, the genotoxicity assays for predicting potential heritable germ cell damage are the same as used for predicting carcinogenicity since the endpoints measured in genotoxicity tests are common precursors for both of these adverse health outcomes. There is a need to evaluate diverse types of biological alterations in order to thoroughly assess the genotoxic/mutagenic potential of a substance; this requires the use of a battery of tests. This section focuses on the role of genotoxicity testing in the prediction of heritable germ cell damage. The use of genotoxicity testing for predicting carcinogenicity is discussed in the section Toxicity Endpoints and Tests: Carcinogenicity.

The classification of germ cell mutagenicity is hazard based, taking into account the intrinsic activity of a chemical to induce genotoxicity in germ cells and is not meant for quantitative risk assessment (UNECE, 2004). Category 1 chemicals are "known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans," and Category 2 chemicals are those "which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans" (UNECE, 2004). Substances that induce heritable damage or damage in germ cells in animals are regarded as suspect human germ cell mutagens as well as possibly having implications for carcinogenicity.

The Animal Test(s)

In vivo tests for assessing potential heritable genotoxicity include:

1. Heritable germ cell mutagenicity tests that include a component that measures damage passed onto progeny are: the mouse heritable translocation test (OECD Test Guideline (TG) 485), the mouse specific locus test, and the rodent dominant lethal test (OECD TG 478).
2. Assays for measuring induction of genotoxicity in germ cells that are used to predict chemicals that might induce damage that is heritable include: the mammalian spermatogonial chromosome aberration test (OECD TG 483), the spermatid micronucleus assay, the mammalian oocyte chromosome aberration/aneuploidy test, and unscheduled DNA synthesis test in testicular cells.
3. Assays for measuring induction of genotoxicity in somatic cells that are used to predict whether a chemical has the potential to induce genotoxicity in germ cells (these are also used in predicting potential carcinogenicity) include: the mammalian erythrocyte micronucleus test (OECD TG 474), the mammalian bone marrow chromosome aberration test (OECD TG 475), the liver unscheduled DNA synthesis (UDS) (OECD TG 486), and the mouse spot test (OECD TG 484) which measures genotoxicity in fetal somatic cells.

In addition to these tests, there are other in vivo methods that are being developed and validated for use in regulatory decisions including the in vivo Comet assay, and in vivo transgenic mutation assays.

Non-animal Alternative Methods

There are a number of in vitro methods for genetic toxicity testing based primarily on bacterial and mammalian cell assays, and several that are accepted by regulatory authorities. Testing proceeds by the use of a tiered test strategy utilizing both in vitro and in vivo assays and providing information for predicting potential heritable germ cell damage as well as potential carcinogenicity.

There are eight in vitro genotoxicity test methods that have been adopted at the EU level with OECD guidelines, four of which are commonly used. These four in vitro assays include two mutagenicity test methods based on bacterial cells: the bacterial reverse mutation test [Ames test], OECD TG 471; and the E. coli reverse mutation assay, OECD TG 472). The two commonly used methods based on mammalian cells are: the in vitro mammalian chromosome aberration test, OECD TG 473; and the in vitro mammalian cell gene mutation test, OECD TG 476. Additionally, the in vitro micronucleus test was validated for genotoxicity testing by ECVAM in 2006 as an alternative to the in vitro chromosome aberration assay (ESAC statement: 17 November 2006; revised OECD TG 487, in preparation).

In addition to these standard in vitro mammalian cell genotoxicity tests, there are in vitro assays for measurement of genotoxicity in primary germ cells but these are not standardized or validated, and there are no ongoing coordinated activities to address this at this time.

Regulatory Requirements & Test Guidelines

Genotoxicity/mutagenicity testing is conducted for pharmaceuticals, industrial chemicals and consumer products, and the results are used to classify chemicals for heritable germ cell mutagenicity as well as carcinogenicity.

Most regulatory agencies and international authorities recommend a test scheme consisting of in vitro and in vivo methods to identify genotoxic/mutagenic substances. A tiered test scheme would likely start with computer-based prediction using quantitative structure-activity relationships (QSAR) and in vitro testing. The International Conference on Harmonization (ICH) recommends a standard core battery for pharmaceuticals, which is undergoing revision at this time.

The working group that was nominated by the European Commission Services to review the status of genotoxicity testing for cosmetics (Maurici et al., 2005) concluded that the above in vivo methods, OECD TG 478, TG 485, TG484, TG483, are not relevant for the purpose of the cosmetic industry.

The UN Globally Harmonized System (GHS) classification of germ cell mutagenicity (UNECE, 2004) utilizes 2 categories: Category 1 chemicals are "known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans." Category 2 chemicals are those "which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans".

Within Category 1, there is category 1A which includes chemicals known to induce heritable mutations in germ cells of humans based on human epidemiological studies. Category 1B includes chemicals that are regarded as if they induce heritable mutations in humans based on positive results in: a) in vivo heritable germ cell mutagenicity tests in animals, b) human germ cell assays for mutagenicity, or c) in vivo somatic cell mutagenicity tests in mammals along with evidence that the substance has potential to cause mutations in germ cells in vivo or information that the substance can interact with germ cell DNA.

Category 2 includes chemicals that are of concern for humans because they have the possibility of inducing heritable mutations in human germ cells. Evidence for classification of a Category 2 chemical is a positive somatic cell mutation test in vivo or other in vivo somatic cell genotoxicity test supported by positive in vitro mutagenicity data. Chemicals that are positive in in vitro mutagenicity tests with structural relationships to known germ cell mutagens should be considered for classification in Category 2.

The GHS recommends use of OECD Test Guidelines for genotoxicity testing, and states that "if new, well validated, tests arise these may also be used in the total weight of evidence to be considered" (UNECE, 2004). The OECD TGs available for in vivo and in vitro genotoxicity and mutagenicity testing are listed in Table 1. The OECD also provides Guidance Document No. 12, Detailed Review Document on Classification Systems for Germ Cell Mutagenicity in OECD Member Countries.

Table 1. OECD Test Guidelines for genotoxicity and mutagenicity testing.

Validation and Acceptance of Non-animal Alternative Methods

In addition to the non-animal alternative methods already accepted and in use (see above), ECVAM has coordinated the retrospective validation of the micronucleus test in vitro, based on existing data. The ECVAM Scientific Advisory Committee (ESAC) endorsed the in vitro micronucleus test (MNT) as a scientifically valid alternative to the in vitro chromosome aberration assay for genotoxicity testing following a weight-of-evidence retrospective validation (ESAC statement: 17 November 2006; draft OECD TG 487). The ESAC statement adds that "the in vitro MNT can then be considered for regulatory use as part of the tier 1 genotoxicity test battery." The draft OECD TG 487 for this method is being revised. An OECD ad hoc expert panel meeting met in Atlanta (October 2007) to finalize the guideline. The meeting was successful and the major issues of concerns were resolved.

ICCVAM has established the Genetic Toxicity Working Group, which is reviewing genetic toxicity test methods. ICCVAM has not validated any new alternative methods for genotoxicity testing at this time.

An ECVAM panel estimated that total replacement of animal testing for genotoxicity/mutagenicity at the EU level would take at least 12 years and require models for evaluating toxicokinetics and metabolism (Maurici, et al., 2005).