July 28, 2020 by Kate Roylance


Genotoxicity testing is required under REACH for substances registered at Annex VII and above. The purpose of genotoxicity testing is to determine whether a substance will cause a mutagenic hazard in humans, that could lead to cancer or cause heritable damage. At Annex VII, an in vitro bacterial-reverse mutation test (OECD 471), investigating gene mutation, is required. At Annex VIII and above, additional in vitro tests are also required investigating cytogenicity (chromosome damage), including a mammalian chromosome aberration test (OECD 473) and a mammalian cell micronucleus test (OECD 487). For genotoxicity testing, in vivo tests are only required if positive results are yielded from the in vitro tests investigating the endpoint in which a positive test occurred.

This means that if a substance produces a positive in vitro result for gene mutation, an in vivo test investigating gene mutation should be conducted, such as the transgenic rodent mutation assay (OECD 488) or the mammalian alkaline comet assay (OECD 489). Also, if a substance produces a positive in vitro result for cytogenicity, an in vivo test investigating cytogenicity should be conducted, such as the micronucleus test (OECD 474) or the chromosome aberration test (OECD 475).

If a substance produces positive in vitro results for BOTH gene mutation and chromosomal aberration, both endpoints must be investigated in vivo. For this purpose, a combined in vivo comet assay (OECD 489) and micronucleus test (OECD 474) can be conducted. This assay will help by providing key information on the gene mutation and chromosomal aberration potential in vivo, and help to reduce animal testing and costs, as it will negate the need to perform two separate in vivo genotoxicity assays.

These tests can be conducted in combination as the micronucleus assay is performed on the peripheral blood cells or bone marrow of rodents, and the comet assay can be performed on a wide array of cells or tissues. The advantages of using the comet assay include its applicability to a wide array of cells or tissues, it’s small requirement of cells per sample, the ease and length of the assay and the relatively low cost of the assay.

Consequently, with careful selection of tissues for investigation, the combination of these assays will allow the detection of potential for chromosome damage in somatic cells of both target tissues relating to the metabolic pathways followed by the test substance, and in circulating blood following systemic exposure.

Recently, ECHA’s Member State Committee has confirmed that a combined in vivo comet assay (OECD 489) and micronucleus test (OECD 474) will be considered as one study. This combined assay can help to reduce animal testing, as highlighted by ECHA in a recent report.

The committee have agreed to request the combined study when:

  • Concerns for BOTH gene mutation and chromosomal aberration have occurred through positive in vitro tests; and
  • No other in vivo genotoxicity data is included in the dossier.

The use of this combined assay will be accepted and will apply to substances in REACH Annex VIII and above, and be subject to compliance checks and testing proposal examinations from 1st July 2020.

If you are interested in performing this test or need help with chemical registrations or the writing of testing proposals, feel free to contact our toxicology department and we can help you.