Toxicity Endpoints & Tests


Last updated: June 20, 2011

Germ cell mutagens/genotoxins are substances that cause heritable (passed on to progeny) changes in the genetic material in germ cells, namely spermatocytes or oocytes (UNECE, 2004; ECVAM, 2002). The term mutagen refers to a substance that induces transmissible changes in DNA structure (Maurici, et al., 2005) involving 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., 2005). The term genotoxicity is generally used unless a specific assay for mutations is being discussed.

At the early testing stages, the genotoxicity assays for predicting potential heritable germ cell damage are the same as used for predicting carcinogenicity because 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 a chemical’s intrinsic ability 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 that “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 animal germ cells 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 genotoxicity induction in germ cells that are used to predict chemicals that might induce heritable damage 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 genotoxicity induction in somatic cells that are used to predict whether a chemical has the potential to induce germ cell genotoxicity (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, other in vivo methods are being developed and validated for use in regulatory decisions, including the in vivo Comet assay and in vivo transgenic mutation assays. For example, JaCVAM is leading an international validation study that includes NICEATM/ICCVAM and ECVAM of the in vivo rodent comet assay, which is being assessed as an alternative for the in vivo rat hepatocyte unscheduled DNA synthesis assay.

Non-animal Alternative Methods

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

Eight in vitro genotoxicity test methods 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, ECVAM coordinated the retrospective validation of the in vitro micronucleus test, based on existing data. The ECVAM Scientific Advisory Committee (ESAC) endorsed the in vitro micronucleus test as a scientifically valid alternative to the in vitro chromosome aberration assay for genotoxicity testing following a weight-of-evidence retrospective validation (ESAC statement, 2006). The ESAC statement comments that “the in vitro [micronucleus test] can then be considered for regulatory use as part of the tier 1 genotoxicity test battery.” The OECD TG 487 for this method was published July 2010.

ICCVAM established the Genetic Toxicity Working Group (GTWG) to review genetic toxicity test methods. The GTWG submitted comments on the draft OECD TG 487, but ICCVAM has not reviewed or endorsed any new genotoxicity test methods 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 [(Q)SAR] 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 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 TGs 478, 483, 484, and 485, are not relevant for the purpose of the cosmetic industry.

The UN Globally Harmonized System (GHS) classification of germ cell mutagenicity (UNECE, 2004) uses two 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 that “cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.”

Within Category 1, category 1A includes chemicals known to induce heritable mutations in human germ cells based on human epidemiological studies. Category 1B includes chemicals 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

TG 471 Bacterial Reverse Mutation Test (Ames Test)
TG 472 Genetic Toxicology: Escherichia coli, reverse assay
TG 473 In Vitro Mammalian Chromosome Aberration Test
TG 474 Mammalian Erythrocyte Micronucleus Test
TG 475 Mammalian Bone Marrow Chromosome Aberration Test
TG 476 In Vitro Mammalian Cell Gene Mutation Test
TG 477 Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster
TG 478 Genetic Toxicology: Rodent Dominant Lethal Test
TG 479 Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells
TG 480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
TG 481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
TG 482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
TG 483 Mammalian Spermatogonial Chromosome Aberration Test
TG 484 Genetic Toxicology: Mouse Spot Test
TG 485 Genetic Toxicology: Mouse Heritable Translocation Assay
TG 486 Unscheduled DNA Synthesis (UDS) Test with Mouse Liver Cells In Vitro
TG 487 In Vitro Mammalian Cell Micronucleus Test
Validation and Acceptance of Non-animal Alternative Methods

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

Combes, R., Grindon, C., Cronin, M.T., et al. (2007). Proposed integrated decision-tree testing strategies for mutagenicity and carcinogenicity in relation to the EU REACH legislation. Altern. Lab. Anim. 35, 267-287.

ECVAM. (2002). Genotoxicity and carcinogenicity. Altern. Lab. Anim. 30, Suppl. 1, 83-93.
Maurici, D., Aardema, M., Corvi, R., et al. (2005). Genotoxicity and mutagenicity. Altern. Lab. Anim. 33, Suppl. 1, 117-130.

Tice, R.R., Agurell, E., Anderson, D., et al. (2000). The single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206-221.

United Nations Economic Commission for Europe (UNECE). (2004).
Globally Harmonized System of classification and labeling of chemicals (GHS). Part 3. Health and environmental hazards. Chapter 3.5. Germ cell mutagenicity. Available here.

Yu, T.W. & Dashwood, R.H. (2007). Measuring antigenotoxic effects using the Ames test and Comet assay. Am. Biotech. Lab. 25, 22-23.