Curren1, R., Mun1, G., Gibson2, D., Aardema2, M. 1Institute for In Vitro Sciences, Inc., Gaithersburg, MD, USA; 2The Procter & Gamble Co., Cincinnati, OH, USA.

This study by scientists at Procter & Gamble Co. and the Institute for In Vitro Sciences (IIVS) demonstrated that MatTek’s EpiDerm in vitro human skin tissue equivalent is an excellent candidate for use in an in vitro micronucleus assay because micronuclei can be reproducibly induced in EpiDerm, the first step in developing a routine “in vivo-like” assay for chromosomal damage in human tissue. 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, Procter & Gamble Co. and IIVS scientists have begun development of a micronucleus assay using a commercially available 3-D engineered skin model of human origin, EpiDerm (MatTek Corp, Ashland, MA). Scientists 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 them 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. 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 0.06-0.6 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.


7th Amendment to the Cosmetics Directive, Binucleated cells, Chromosomal damage in human tissue, Commercially available, Cyt B, Cytochalasin B (Cyt B), EpiDerm, European legislation, Genetic toxicology, Human 3-D skin model, Human origin, MMC, Micronuclei formation, Micronuclei induction, Micronucleus assay, Mitomycin C (MMC), Non-animal test methods, Point of contact, Reproducibly induced, Rodent in vivo micronucleus assay, Routine in vivo-like assay, Tiered testing strategy, Topical application protocol, Toxicity

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