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The working group nominated by the European Commission Services to review the status of genotoxicity testing for cosmetics concluded that the following issues are not sufficiently addressed with current in vitro methods for genotoxicity/mutagenicity testing: the effect of human metabolism; toxicokinetics; the use of cell lines relevant to the target organs; and the oversensitivity (false positives) of in vitro assays (Maurici, et al., 2005). The panel proposed a four-stage test strategy to characterize substances for the cosmetics industry, where Stage 3 is a new stage:

• Stage 1: Evaluation of existing data and knowledge
• Stage 2: Use of an in vitrotest battery of currently accepted methods for hazard identification
• Stage 3 (new): Use of target in vitromodel systems (methods still need to be developed and validated) for substances testing positive in any stage 2 test. Because skin cells are the site of first contact for most cosmetics and many industrial chemicals, skin has a high level of exposure, and the following Stage 3 tests are proposed: the Comet assay in primary skin cells or models; a micronucleus test in primary skin cells or skin models (if chromosomal aberrations or micronuclei are induced in Stage 2). For photo-mutagenicity/genotoxicity, similar tests can be considered. If the results of the tests performed in stage 3 are negative, further testing, including testing for germ cell effects, should not be necessary. For nondermal cosmetics, new tests involving appropriate primary cultures or models would need to be developed and validated for the assessment of mutagenicity/genotoxicity.
• Stage 4: Use of in vivo tests for substances testing positive in any stage 3 test.

The proposed approach provides several options for avoiding/decreasing animal testing, such as not requiring further testing on substances that are not absorbed through the skin.

The Comet assay, both in vitro and in vivo, is being developed and validated for regulatory purposes. Recommendations for standardizing protocols were published (Tice, et al., 2000), and an ECVAM panel provided additional recommendations for completing validation (Maurici, et al., 2005, p. 123). The Japanese Center for the Validation of Alternative Methods (JaCVAM) is leading the validation of the Comet assay. The Comet assay involves an electrophoretic method to detect DNA strand breaks and alkaline-labile DNA lesions in cells. It is commonly used for eukaryotic cells owing to its versatility (Yu & Dashwood, 2007). Very small cell samples can be analyzed using an agarose gel “sandwich” on a microscope slide and microscopic visualization of the ethidium bromide-stained DNA migration. The Comet assay is very sensitive, can be used as an alternative to other less sensitive in vivo tests for DNA damage (like the unscheduled DNA synthesis assay), and can be used on cell lines, primary cell cultures, and in vivo tissues (Maurici, et al., 2005).

Other in vitro methods for genotoxicity/mutagenicity testing are in various stages of development and validation, yet assays specifically to address effects in germ cells are not a current focus.

An integrated testing scheme for genotoxicity and carcinogenicity has been proposed to address the anticipated increase in testing requirements under the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system (Combes, et al., 2007). “This scheme makes maximum use of pre-existing data, computer (in silico) and in vitro methods, with weight-of-evidence assessments at each major stage. The need for the improvement of in vitro methods, to reduce the generation of false-positive results, is also discussed.”

Additional information on these areas of emerging research, methods, and policies will be provided on AltTox.org in the future; please check back again. Also, please check the commentaries in the Way Forward section.