Category: Technical References, EpiDerm
427. FURTHER DEVELOPMENT OF A MICRONUCLEUS ASSAY USING THE HUMAN 3-D SKIN MODEL, EPIDERM™.
Curren1, R.D., Aardema², M.J., Hayden³, P.J., Mun1, G., Hu², T. 1Institute for In Vitro Sciences, Gaithersburg, MD, ²Procter & Gamble, Cincinnati, OH, ³MatTek Corporation, Ashland, MA. Presented at the 14th International Workshop on In Vitro Toxicology, Ostend, Belgium, October 2-5, (2006).
Summary:
This study by scientists at Procter & Gamble Co. and the Institute for In Vitro Sciences (IIVS) demonstrated that MatTek’s EpiDerm human skin tissue equivalent shows promise as an in vitro human skin micronucleus assay for genotoxicity testing of cosmetic ingredients.
Scientists at Procter & Gamble Co. and the Institute for In Vitro Sciences (IIVS) are developing an in vitro reconstructed human skin micronucleus assay (RSMA) using the 3D EpiDerm™ skin model (MatTek Corp, Ashland, MA) for potential use in testing cosmetic ingredients as a replacement for in vivo genotoxicity assays that will be banned starting in 2009 according to the EU 7th Amendment to the Cosmetics Directive.
The EpiDerm model was chosen because it theoretically better approximates the complexities of in vivo dermal exposures and reflects human-specific responses.
The standard protocol utilizes two 10 ul topical doses of test material given 24 hours apart in the presence of 3 ug/ml cytochalasin B in medium. Cells are harvested from the tissue 24 hours after the last dose.
The background frequency of micronuclei is reproducibly low at <0.2%, with a statistically significant, reproducible positive responses around 1.2% (3 ug/ml MMC) and 2.3% (6 ug/ml MMC). Previously, it has been shown that dose-related increases occur in both cytotoxicity and micronuclei induction for several model genotoxins, e.g. MNNG, MMS and vinblastine sulfate.
The assay was further evaluated with 4 model non-carcinogens as determined in dermal bioassays: trichloroethylene, 4-nitrophenol, 2-ethyl-1,3-hexanediol, and 1,2-epoxydodecane. All four model non-carcinogens in rodent skin are negative in the in vitro RSMA. These results have further demonstrated the potential utility of such an assay.
Dermal biotransformation capabilities of the EpiDerm in vitro human skin model have been assessed. Enhanced metabolism of the CYP1A1 and CYP1B1 substrate, ethoxyresorufin, has been shown following treatment with 3-methylcholanthrene (3-MC) and â-naphthoflavone (â-NF). GST and UDP glycosyltransferase activity was also evaluated by measuring conjugation of glutathione with 1-chloro-2,4-dinitrobenzene, andUDP with 4-methylumbellipherone, respectively. N-acetyltransferase 1 activity was evaluated by measuring the acetylated metabolites of para-aminophenol (PAP) and paraphenyldiamine (PPD).
The results demonstrate that the EpiDerm skin model possesses numerous human in vivo-like metabolic activities and thus will likely be useful for evaluating chemicals that require dermal biotransformation. This novel assay system continues to hold excellent promise as a human “in vivo-like” genotoxicity model.
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EpiDerm Data Sheet
EpiDerm Specifications
EpiDerm Technical References
Applications: 7th Amendment Cosmetics Directive, Cosmetics analysis, Genotoxicity, Metabolism - skin, Metabolism - xenobiotic, Micronucleus assay, REACH
Keywords: 7th Amendment to the Cosmetics Directive, Binucleate Cells, Cosmetic Directive, Cosmetics, Cytochalasin B, EU 7th Amendment, Epiderm, Genotoxicity, Genotoxicity tests, Histology, In vitro, In vivo, Metabolism, Micronucleus, Micronucleus assay, Photogenetox, REACH, REACH program, RT-PCR, Stratum granulosum, Vinblastine sulfate (VB), Xenobiotic metabolizing activity
Endpoints: Genotoxicity tests
Materials Tested: 1,2 Epoxydodecane, 2-ethyl-1,3-hexanediol, 4-Nitrophenol, BP, CP, DMBA, MMC, MMS, MNNG, Trichloroethylene, VB
