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Human 3D Skin Models

Wed, 04/10/2013 - 1:36pm
Scott Hickman, Marketing Manager, Toxicology, BioReliance Corporation

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A Non-animal Based Method for Genotoxicity Testing of Chemical Compounds and Cosmetic Ingredients

When developing a new chemical compound or cosmetic ingredient, a series of tests may be performed to show that the item is not harmful to health. In the past, some of this testing has been done using animals; this testing is referred to as in vivo. However, testing for genotoxicity (damage to a cell's genetic material affecting its integrity) of cosmetic ingredients on animals was banned in Europe in 2009, under the seventh amendment to the EU Cosmetics Directive, and since then there has been significant effort undertaken to find new ways to safely and effectively test cosmetics ingredients used in topical applications. In addition, there is an increased emphasis within industry, regulatory, and academic groups as a whole on the use of non-animal based methods for the safety testing of chemicals and drugs.

Finding effective methods for genotoxicity testing without in vivo models has proven to be challenging. Existing in vitro tests may result in false positives, so a battery of specific in vitro genotoxicity tests are generally followed by additional in vivo tests on those test articles (chemical compounds) that gave a positive result. This approach is performed to exclude false positives and help to establish genotoxic potential early on in the product development process. This also allows manufacturers to avoid continued research efforts into ingredients that could ultimately prove unsafe.

The EU ban on animal testing for cosmetics has had major repercussions because the follow-up in vivo tests used in the past to address false positive results are, quite simply, no longer allowed. Although no such ban yet exists in the US, the global nature of the cosmetic and personal care business means that in order to do business in EU-regulated markets, US companies are now required to adhere to this regulation as well. Because of this, researchers have been keen to identify an in vitro test that could factor in many of the parameters that are at play in live animal skin while avoiding high levels of false positives.

One of the delineaters in the researcher's development was to evaluate with CREST-labeling and FISH to understand mechanism (whether aneugenic or clastogenic).The picture shows CREST-labeled cells showing both a normal result and a positive micronucleus result, thus showing the Test Article(TA) to be aneugenic.The micronucleus assay is one of the most common assays for measuring chromosomal damage (a genotoxicity measure). Based on directly treating cells in culture with a test article, it detects extra nuclei in the cell cytoplasm that represent chromosomal fragments (or even whole chromosomes) that have been excluded from the nuclei during cell division. It is an accepted technique for a genotoxicity testing series and is common in regulatory submissions. However, traditional in vitro cell cultures do not account for the 3D structure of the skin and the way topical application of an ingredient might affect the different skin layers.

The development of the 3D reconstructed human skin micronucleus (RSMN) assay is the first to overcome the limitations of traditional cell culture methods. It can provide a more biologically relevant result than standard 2D in vitro genotoxicity assays, since it provides a functional stratum corneum, which accounts for permeability and appears to have a normal dermal metabolic capability. It is also expected to have more normal DNA repair and cell cycle control, unlike the p53 deficient cell lines that are used in the majority of standard in vitro genotoxicity assays.

The 3D skin model used in the BioReliance GLP RSMN test is supplied by MatTek and consists of normal, human-derived epidermal keratinocytes from human foreskin. The cells are cultured to form a multi-layered, highly differentiated model of the human epidermis, which closely resembles normal human skin. Recent studies and publications have shown that the RSMN test is more predictive than traditional in vitro cell cultures.

Qualification studies of this new 3D Skin assay conducted by BioReliance with chemicals that have numerous established modes of action, including DNA cross-linking agents, aneugens and clastogens, show dose-dependent increases of micronuclei. Importantly, chemicals that are not hazardous produced negative results in the RSMN assay. The tests demonstrated good reproducibility, and the results were comparable to those previously published.

To meet industry interest in understanding mechanisms of genotoxicity, the RSMN assay was further developed to incorporate centromere labelling of micronuclei for the identification of aneugenic versus clastogenic mechanisms. Aneugenicity is a change in the number of chromosomes in a cell as a result of disruption of the cellular machinery involved in segregating chromosomes. Clastogenicity is a result of chromosome breakage. For example, the anti-microtubule cancer drug vinblastine sulfate induces a large increase in micronuclei with positive centromere labelling indicative of aneugenicity, as expected for a drug with this mechanism of action.

The availability of the 3D Skin (RSMN) test offers an improved approach for genotoxic testing of skin exposures.  Utilization of this assay will allow the cosmetic industry (as well as other chemical based manufacturers) to continue reducing or eliminating the need for animal based tests.


a FISH result, showing an aneugenic mechanism for the test article.Potential Use of RSMN In Other Sectors including Pharmaceuticals

While the cosmetic sector has pioneered the use of the 3D skin model because of the ban on animal testing, it has great potential in other sectors where animals are still being used for genotoxicity testing. Since skin is often the tissue with the highest exposure to industrial chemicals, agrochemicals, and some drugs, the RSMN assay is an ideal approach for a variety of sectors. Although the assay is conducted using skin (in culture), it can be considered a surrogate for other highly exposed tissues.

Though there is growing interest within the pharmaceutical industry to reduce animal tests, it will continue to prove more difficult to achieve for drugs that are designed to be ingested, and are thus much more likely to interact with internal organs such as the liver and the kidneys. Regulatory bodies still require potential drugs to be extensively tested on animals before they are allowed to be used in human subjects in clinical trials, including tests to predict genotoxic potential. Despite the extra challenge, there have been real moves toward reducing the number of animals used in testing and replacing them with in vitro alternatives where possible. The use of the RSMN assay for dermal drugs is an approach consistent with this goal.

The GLP RSMN skin test represents a paradigm shift toward the use of more biologically relevant 3D models for genotoxicity testing and safety testing in general. This test is sure to be the first of many 3D models that will be developed for assessing safety. There are numerous 3D models available for adaptation into safety tests, and it is easy to envision the technique expanding to other organ tissue types, such as liver and kidney; which are particularly important tissues for the pharmaceutical industry, or an airway model; useful for safety testing within the chemical industry.

The development of safety tests in 3D models is a critical step towards the goal of employing more biologically relevant assays for screening and testing drugs and chemicals, as well as greatly contributing to a reduction in the use of animals for testing. This is not only the future of genetic toxicology, but for the pharmaceutical industry as a whole.

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