The Cosmetics Regulation (EC) No 1223/20091 is the main regulatory framework for finished products when placed on the EU market.
Cosmetic products must be safe for human health and the regulation requires that they undergo a safety assessment. The safety of cosmetic products is based on the safety of the ingredients2,3 and their toxicological profile is evaluated.
The determination of the toxic potential of a cosmetic substance is based on a series of toxicity studies and forms part of the hazard identification.
The major endpoints that may be relevant for the toxicological profile are the following: eye and skin irritation, skin sensitization, photo-induced toxicity, skin penetration and genotoxicity. For each one, different test methods may be used.
Among these toxicological parameters genotoxicity is one of the critical endpoints.
Genotoxicity refers to all phenomena that alter the genetic material of a cell or its transmission. Hence different processes could occur:
- Mutagenicity. This is a change in the nucleotide sequence in the DNA. This leads to a codon modification or a change in the DNA reading frame and consequently to a single gene or a gene segment modification. These changes could be transferred to the next generation.
- Clastogenicity. This is a structural modification of the DNA such as gaps, breakages or chromosome rearrangements.
- Aneugenicity. This is an effect that leads to a change in the number of chromosomes due to a loss a gain of one or more chromosomes at the time of their separation during the anaphase step of the mitosis.
Thus, genotoxicity evaluation of a compound must include tests that can detect these three endpoints, i.e. mutagenicity at the gene level, chromosome breakage and/or rearrangements (clastogenicity), and numerical chromosome change (aneugenicity).
In addition, since March 11, 2013, a complete ban on animal testing for cosmetic products is implemented in the EU. The ban applies to all new cosmetics and their ingredients sold in the EU, regardless of where in the world testing on animals was carried out. Since this date, no animal testing can take place in the EU and no company can carry out new animal tests for cosmetics purposes outside of the EU for products to be sold in the EU. The Cosmetic Regulation 1223/2009 contains the same provisions (Article 18).
Tests should be performed according to the OECD test guidelines and four in vitro tests have been validated to assess the 3 endpoints, with test guidelines (TG):
|In vitro tests||Endpoints||OECD|
|Bacterial reverse mutation test (Ames test)||Gene mutations||TG471|
|Mammalian cell gene mutation test / Mouse Lymphoma Assay (MLA)||Gene mutations||TG490|
|Mammalian chromosomal aberration test||Structural aberrations||TG473|
|Mammalian cell micronucleus test||Structural and numerical aberrations||TG487|
Due to the fact that no individual test can cover all the 3 endpoints, they should not be used as stand-alone tests. A battery of tests, part of testing strategy, must therefore be used in order to provide the adequate coverage of the endpoints.
The SCCS recommends two tests3 for the base level testing of cosmetic substances, represented by the following test systems:
- Ames test (OECD 471) as a test covering gene mutations
- In vitro Micronucleus test (OECD 487) as a test for both structural (clastogenicity) and numerical (aneugenicity) chromosome aberrations.
Four scenarios possible are presented in the Addendum to the SCCS's Notes of Guidance, 8th Revision4 and, if needed, further strategies are proposed.
If both tests (i.e. Ames and Micronucleus) are negative, it is very likely that the substance has no mutagenic potential. If both tests are positive, unless a weight of evidence (WoE) approach can overruled this fact, it is very likely that the substance has mutagenic potential. In both cases, no further testing is necessary.
If one test is positive, the substance is considered as in vitro mutagen and further testing can be used to better assess the mutagenic potential. If the Ames is positive and the Micronucleus negative, a MLA (OECD 490) test will be preferred. In the opposite case, the SCCS recommends either a comet assay or a Micronucleus test, both in the 3D-reconstructed human skin model. The Mammalian chromosomal aberration test (OECD 473) can also be considered.
The genotoxixity tests in the 3D-reconstructed human skin model have the advantage of both making a bridge between in vitro and in vivo tests and leading to allow the assessment of poorly soluble compounds.
In any case, a weight of evidence (WoE) approach has to be considered if the previous strategy does not lead to a clear answer. A positive in vitro result in genotoxicity testing is indicative for the carcinogenic potential of substances.
Whatever your project or your product is, the genotoxic potential of an ingredient is a critical element of the safety assessment and IDEA Lab can help you to establish the right testing strategy according to the type of sample and in combination with the other toxicological tests.
References:1. Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products (Cosmetics Regulation).
2. Guidelines on Annex I to Regulation (EC) No 1223/2009 of the European Parliament and of the Council on cosmetic products, November 25, 2013 (2013/674/EU)
3. SCCS’s Note of guidance for the testing of cosmetic ingredients and their safety evaluation, 9th revision, September 29, 2015 – revised April 2016 (SCCS/1564/15)
4. Addendum to the SCCS's Notes of Guidance (NoG) for the Testing of Cosmetic Ingredients and their Safety Evaluation, 8th Revision (SCCS/1501/12)
Frédéric NUNZI, PhD
Doctor in Cell Biology and Microbiology / Head of IDEA Lab
Eurotox Expert (Regulatory and in vitro Toxicology)