Development of a Novel Micronucleus Assay in the Human 3-D Skin Model, EpiDerm TM . R D Curren 1 , G Mun 1 , D P Gibson 2 , and M J Aardema 2 . 1 Institute for In VitroSciences, Inc., Gaithersburg, MD; 2 The Procter & Gamble Co., Cincinnati, OH. Presented at the 44nd Annual Meeting of the Society of Toxicology New Orleans, Louisiana March 6-10, 2005 Abstract The rodent in vivo micronucleus assay is an important part of a tiered testing strategy in genetic toxicology. However, this assay, in general, only provides information about materials available systemically, not at the point of contact, e.g. skin. Although in vivo rodent skin micronucleus assays are being developed, the results will still require extrapolation to the human. Furthermore, to fully comply with recent European legislation
such as the 7th Amendment to the Cosmetics Directive, non-animal test methods will be needed to assess new chemicals and ingredients. Therefore we have begun development of a micronucleus assay using a
commercially available 3-D engineered skin model of human origin, EpiDerm TM (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 (to at least 120 h) and with increasing concentration of Cyt B. Three ug/ml Cyt B allowed us to reliably obtain 40-50% binucleated cells at 48h. Mitomycin C (MMC) was then used (in the presence of 3 ug/ml Cyt B) to investigate toxicity and micronuclei formation in EpiDerm TM . Exposing the tissue directly through the growth medium for 48h gave a dose response for toxicity between 0.03 and 0.6 ug/ml. Maximum micronuclei induction (~5%) occurred at 0.1-0.3 ug/ml MMC. Experiments conducted with and without Cyt B indicated higher frequencies in the presence of Cyt B as expected. A topical application
protocol was then developed using two 10 ul (per 0.64 cm2 tissue) applications of MMC in ethanol 24 and 48h prior to harvest. Maximum micronucleus response (~8%) and toxicity occurred with applications of 6-60 ug/ml MMC. The background frequency of micronuclei was very low (~0.1%). These studies show that
micronuclei can be reproducibly induced in a 3-D skin model and are the first steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. Introduction The in vitro micronucleus assay has been well established for use in detecting potentially clastogenic and aneugenic chemicals (1, 2). Its acceptance has been especially helped by the development of the
cytokinesis-block methodology (3) which allows the identification of cells which have undergone one nuclear division, and modulates differences in cell division kinetics due to cytotoxicity, growth conditions or cell origin. However, because there is a need to know whether chemicals which are active in vitro are also
bioavailable and active in the whole organism using conventional exposure techniques, in vivo micronucleus techniques have been developed and standardized for several organ/tissue systems, but most often use those of the hematopoietic system (e.g. 4). Recently an interest has developed in utilizing a tissue, skin, that is often a target organ for human exposure, both from natural substances and consumer products (5-8). However, these methods generally
use either the rat or mouse and may not be completely reflective of what would occur in humans. In addition, new regulations in Europe such as the 7th Amendment to the Cosmetic Directive and the REACH program,
will make it very difficult, and in some cases impossible, to use animal models in safety assessments. Therefore we have begun investigations to determine if human three-dimensional skin constructs can be used as a target system for micronucleus studies. Theoretically such models could not only reflect the
complexities typical of in vivo exposures, e.g. absorption, tissue specificity, metabolism, etc., but at the same time reflect human-specific responses in these parameters. We have used a commercially available skin model, EpiDerm TM (MatTek Corp., Ashland, MA), to begin our studies with the model genotoxicants, mitomycin C (MMC) and vinblastine sulfate (VB). We show here that the background frequency of micronuclei is very low and reproducible, and that high frequencies of micronuclei can be induced by both topical exposure and exposure through the medium. Materials & Methods Test Chemicals Mitomycin C, Sigma (St. Louis, MO) Vinblastine Sulfate, Sigma (St. Louis, MO) Reagents Acetone, Aldrich Chemical Co. Acridine Orange Solution (10 mg/ml), Sigma, (final concentration 40