Regulatory safety assessment

Safety assessment of new chemical entities to which humans will be exposed is regulated and as such follows guidelines issued by industry sector-specific regulatory bodies. The central tenet of safety assessment genotoxicity testing is an assay battery approach comprised of the following

  1. In vitro – a bacterial reverse gene mutation test
  2. In vitro – a mammalian cell test for chromosomal damage (mutation, chromosome aberrations, or micronuclei formation)
  3. In vivo – micronuclei formation (in blood or bone marrow) or chromosome aberrations in bone marrow

The above assay battery is deployed in different ways according to the industry sector, following specific obligations / legislation. For example, the European Cosmetics Directive 76/768, 7th Amendment prohibits the use of animal testing for finished cosmetics products, ingredients or combinations of ingredients, whilst the recently revised ICH guidance for pharmaceuticals intended for human usage (S2R1) allows 2 options, either combining all 3 of the above battery elements or performing the first and third but with the addition of another in vivo endpoint.

Key to the performance of safety assessment genotoxicity tests are the published OECD Guidelines for the Testing of Chemicals. These test-specific guideline documents provide descriptions of internationally agreed methodologies that are referenced by cross-sector regulatory bodies.

In vitro – Ames bacterial reverse gene mutation. (OECD 471)

This test employs strains of Salmonella typhimurium and Escherichia coli that require specific amino acid supplementation, to detect point mutations. In these testing strains, such mutations result in the restoration of the bacterial cells’ capability to synthesize an essential amino acid, and hence grow in the absence of the amino acid.

In vitro – Mammalian cell tests for chromosomal damage.

Micronucleus test (OECD 487): this test detects micronuclei in the cytoplasm of interphase cells, which may originate from acentric chromosome fragments or whole chromosomes that have mis-segregated during anaphase. Both aneugenic and clastogenic chemicals are detected in cells that have undergone cell division after chemical exposure.

Chromosome aberration assay (OECD 473): this test is used to microscopically detect structural chromosome aberrations in cells that have been arrested in metaphase after chemical treatment.

In vitro – Gene mutation (mouse lymphoma assay; (OECD 476)

The thymidine kinase (TK) endpoint is the most commonly used, though hypoxanthine-guanine phosphoribosyl transferase (HPRT) and xanthine-guanine phosphoribosyl transferase (XPRT) are also used. Mutation of TK-heterozygous cells to TK-deficient cells results in the capability to grow in the presence of a cytotoxic pyrimidine analogue, which TK-proficient cells are sensitive to.


In vivo – micronuclei formation or chromosome aberrations.

Mammalian erythrocyte micronucleus test (OECD 474): this test is used to detect micronuclei in erythrocytes sampled from the peripheral blood or bone marrow of treated animals (usually rodents).

Mammalian bone marrow chromosome aberration test (OECD 475): this test is used to microscopically detect structural chromosome aberrations in bone marrow cells taken from animals (usually rodents) that have undergone chemical treatment followed by metaphase arrest.