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DEVELOPMENT OF A MICRONUCLEUS ASSAY IN THE EPIDERM™ HUMAN 3D SKIN MODEL.
Curren1, R., Mun1, G., Gibson2, D., and Aardema2, M.
1Institute for In Vitro Sciences, Inc., Gaithersburg, MD; 2The Procter & Gamble Co., Cincinnati, OH.
Presented at the Environmental Mutagen Society Meeting, Pittsburgh, PA. October 2-6, (2004).
Keywords: Absorption, Acetone, Aneugenic chemicals, Basal layer, Binucleate, Calcium/magnesium-free dulbecco’s phosphate buffered saline (DPBS), Clastogenic chemicals, Cosmetics, Cytochalasin B, Cytokinesis blockage, DMSO, DPBS, EPI-100-NMM, EU 7th Amendment, Epiderm, Epiderm tissue, Ethanol, Genotoxicants, Genotoxicity tests, Histology, Keratinocytes, Kinetics, MMC, Metabolism, Micronucleus assay, Micronucleus studies, Mineral oil, Mitomycin C (MMC), NMM, REACH program, Safety, Safety assessments, Saline, Solvents tested, Stratum corneum, Stratum corneum barrier, Three-dimensional skin constructs, Tissue specificity, Topical dosing, Toxicity, Trypsin, VB, Vinblastine sulfate (VB)
Endpoints: Genotoxicity tests
Materials Tested: 10 ul EtOH, 10 ul Saline, 10 ul acetone, 10 ul acetone:oil (4:1), 20 ul EtOH, 20 ul acetone, 20 ul acetone:oil (4:1), Acetone, Ethanol, Saline
Summary: To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetic ingredients will not be allowed. Skin – because of its generally high exposure – is a target area of interest for many cosmetic products. It would be beneficial to have a skin-based micronucleus assay that did not require live animals, and even more appropriate if it utilized human skin in vitro. We describe preliminary work on a human tissue-based micronucleus assay using EpiDerm™ engineered human skin (MatTek Corp., Ashland, MA). Since little is known about the kinetics of the dividing keratinocytes in the EpiDerm™ model, we evaluated exposure to 1, 2 or 3 µg/ml cytochalasin B for varying lengths of time to determine whether a reasonable population of binucleated cells for development of a micronucleus assay could be obtained. The frequency of binucleated cells increased both with time (to at least 120 h) and with increasing concentration of cytochalasin B. Using medium NMM, which supported the differentiated state of the model through 48 h, 40 to 50% binucleated cells were reliably obtained at 48 h, making this a convenient time point for subsequent experiments. We evaluated different solvents and determined that up to 20 µl (per 0.62 cm2 tissue) of ethanol, acetone, and a mixture of acetone: mineral oil had no impact on the frequency of binucleated cells, whereas the frequency of binucleated cells was decreased with DMSO or higher volumes of saline. Exposure of the cultures to vinblastine sulfate or mitomycin C topically or through the media, resulted in toxicity as evidenced by a dose dependent reduction in the percentage of binucleated cells.
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