THE RECONSTRUCTED SKIN MICRONUCLEUS ASSAY (RSMN) IN EPIDERM™: DETAILED PROTOCOL AND HARMONIZED SCORING ATLAS.

Dahl1, E.L., Curren1, R., Barnett2, B.C., Khambatta2, Z., Reisinger3, K., Quedraogo4, G., Faquet4, B., Ginestet4, A-C., Mun1, G., Hewitt5, N.J., Carr2, G., Pfuhler2, S., Aardema6, M.J. 1Institute for In Vitro Sciences, Inc., Gaithersburg, MD, USA. 2Procter & Gamble, Miami Valley Laboratories, Cincinnati 45253, USA. 3Henkel AG & Co KGaA, Duesseldorf, Germany. 4L'Oreal Life Sciences Research, Aulnay sous Bois, France. 5SWS, Erzhausen, Germany. 6Marilyn Aardema Consulting, LLC, 5315 Oakbrook Dr., Fairfield, OH, USA.

Abstract

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.

Keywords

Acridine-orange staining, Bacterial contamination, Bi-nucleated cells, Cell harvest, COLIPA, EpiDerm™, Genotoxicity, Interlaboratory transfer, Micronuclei, Micronucleus, Protocol, Reconstructed skin, Reconstructed skin micronucleus (RSMN) assay, Scoring, Solvents

Materials Tested

Acetone, Acetone:olive oil 4:1, Benzo(a)pyrene, Cytochalasin B, DMSO, DMSO 50%, DMSO 75%, Ethanol, Ethanol 75%, Ethanol 90%, Mitomycin C, Saline

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