The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.
The in vivo comet assay (single cell gel electrophoresis assay) in its alkaline version (pH >13) is being increasingly used in genotoxicity testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals. Recommendations for an appropriate performance of the test using OECD guidelines for other in vivo genotoxicity tests have been published. In this review, we critically discuss the biological significance of comet assay effects in general and the status of the test in current strategies for genotoxicity testing. Examples for practical applications of the in vivo comet assay and potential consequences of positive and negative test results are given. The significance of comet assay results for hazard identification and risk assessment is discussed. In accordance with international guidelines for genotoxicity testing the in vivo comet assay is recommended for follow-up testing of positive in vitro findings. It is particularly useful as a tool for the evaluation of local genotoxicity, especially for organs/cell types which cannot easily be evaluated with other standard tests. A positive result in an appropriately performed in vivo comet assay indicates genotoxicity of the test compound in the tissue tested and gains particular significance when a mutagenic potential of the test compound has already been demonstrated in vitro. Such findings will have practical consequences in the risk assessment processes and further development of substances.
Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.
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