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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 is one of the target areas of interest for many cosmetic products because it is generally the tissue with the highest exposure. Therefore we have begun development of a micronucleus assay using a commercially available 3-D engineered human skin model, EpiDerm™ (MatTek Corp, Ashland, MA). We first evaluated whether a population of binucleated cells sufficient for a micronucleus assay could be obtained by exposing the tissue to 1-3 ug/ml cytochalasin B (Cyt B). The frequency of binucleated cells increased both with time and with increasing concentration of Cyt B. Cyt B at 3 ug/ml allowed us to reliably obtain 40–50% binucleated cells at 48 h and was used in future studies. The background frequency of micronuclei in this model is low (~0.1%) and reproducible. Studies with model genotoxins including mitomycin C, vinblastine sulfate and methylmethane sulfonate demonstrated that micronuclei can be reproducibly induced in this 3-D skin model. This is the first step in developing a routine “in vivo-like” assay for chromosomal damage in human tissue.