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(프로토콜) Skin Irritation (ECVAM Validated)
 

 
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Skin Irritation (ECVAM Validated) 

 

The Model

MatTek's patented EpiDerm™ System consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. These "ready-to-use" tissues, also known generically as Normal Human-3D (NHu-3D), are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation on the cutting edge of in vitro skin technology. Ultrastructurally, the EpiDerm Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology. For more information on the EpiDerm model, click here.

The Method

프로토콜 요청은 이곳으로.

EpiDerm Skin Irritation Test Training 동영상 보기는 이곳에서.

 

  1. Transfer tissues from agarose to assay medium

  2. Incubate (37 ±1°C, 5 ±1% CO2, 95% RH) for 60 ±5 min

  3. Transfer tissues to fresh assay medium

  4. Incubate (37 ±1°C, 5 ±1% CO2, 95% RH) overnight (18 ±3 h)

  5. Dose 3 tissues each with 30 μl/25 mg TS, PC, or NC

  6. Expose 60 min (37 ±1°C, 5 ±1% CO2, 95% RH)

  7. Stop exposure after 60 minutes by rinsing in DPBS

  8. Transfer tissues to fresh assay medium

  9. Incubate (37 ±1°C, 5 ±1% CO2, 95% RH) for 24 ±2 hours

  10. Collect medium for IL-1α analysis (optional)

  11. Incubate (37 ±1°C, 5 ±1% CO2, 95 % RH) for 18 ±2 hours

  12. Blot tissues and perform MTT assay

  13. Read OD in a plate spectrophotometer at 550-570nm

 

The Endpoints

MTT Tissue Viability Assay

IL-1α (optional)

 

Regulatory Acceptance

European Centre for the Validation of Alternative Methods (ECVAM)

OECD TG 439

 

Technical References

 

697. EVALUATION OF AN IN VITRO HUMAN SKIN IRRITATION TEST FOR USE WITH MEDICAL DEVICE EXTRACTS. Coleman1, K.P., Casas1, J.W., Lewerenz1, G.M., Rankin1, E.A., Willoughby2, J.A. Sr., Blakeman2, L.C. and McKim2, J.M. Jr. 1Medtonic, Inc., Minneapolis, MN, 55432 2CeeTox, Inc., Kalamazoo, MI, 49008

693. CLASSIFICATION AND LABELING OF INDUSTRIAL PRODUCTS WITH EXTREME PH BY MAKING USE OF IN VITRO METHODS FOR THE ASSESSMENT OF SKIN AND EYE IRRITATION AND CORROSION IN A WEIGHT OF EVIDENCE APPROACH. Scheel1, J., Heppenheimer2,A., Lehringer1, E., Kreutz1, J., Poth2, A., Ammann1, H., Reisinger1, K., Banduhn1, N.  1Henkel AG & Co. KGaA, Düsseldorf, Germany. 2Harlan Cytotest Cell Research GmbH, Rossdorf, Germany. Toxicology in Vitro, 25, 1435–1447 (2011).

683. UPDATE ON VALIDATION STATUS AND INDUSTRY UTILIZATION OF NORMAL HUMAN 3D (NHU-3D) ANIMAL ALTERNATIVE MODELS. Kandarova, H., Klausner, M., Kubilus, J., Ayehunie, S., Hayden, P., Kaluzhny, Y., Letasiova, S., and Sheasgreen, J.  MatTek Corporation, Ashland, MA, USA and MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Presented at 8th World Congress on Alternative and Animal Use, Montreal, Canada, 2011

657. IN VITRO ASSESSMENT OF SKIN IRRITATION POTENTIAL OF SURFACTANT BASED FORMULATIONS USING 3-D SKIN RECONSTRUCTED TISSUES AND CYTOKINE EXPRESSION ANALYSIS. Gandolfi1, L., Tierney1, N., Johnson1, D., Walters1, R., Fevola1, M., Gunn1, E., Martin1, K., Kong2, A., Hilberer2, A., Barnes2, N., Wilt2, N., Nash2, J.R., Inglis2, H., Raabe2, H., Costin2, G-E. 1Johnson & Johnson, Skillman, NJ, United States 2Institute for In Vitro Sciences, Inc., Gaithersburg, MD, United States. Presented at SOT 2011, Abstract #2555

649. IL-18 SECRETION AS A MARKER FOR IDENTIFICATION OF CONTACT SENSITIZERS IN THE EPIDERM IN VITRO HUMAN SKIN MODEL. Deng, W., Oldach, J., Armento, A., Ayehunie, S., Kandarova, H., Letasiova, S., Klausner, M., and Hayden, P. MatTek Corporation, Ashland, MA, USA. Presented at SOT 2011, Abstract #2571

622. MEETING REQUIREMENTS OF OECD TG 404 FOR IN VITRO SKIN IRRITATION TESTING: REPRODUCIBILITY OF EPIDERM SKIN IRRITATION TEST (EPIDERM-SIT) FOLLOWING ECVAM VALIDATION AND ACCEPTANCE AS A FULL REPLACEMENT METHOD. MatTek Corporation, Ashland, MA, U.S.A. IIVS In vitro Alternatives 2010 Forum.

583. NON-ANIMAL METHODS FOR TOXICITY TESTING: THE WAY FORWARD FOR IN VITRO SKIN IRRITATION TESTING. Hayden, P. MatTek Corporation. AltTox.org.

582. NON-ANIMAL METHODS FOR TOXICITY TESTING: SKIN IRRITATION/CORROSION. AltTox.org, 2008

 

  

 

 

 

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