The EpiOcular™ Model Protocol, Performance and Experience
Transcripción
The EpiOcular™ Model Protocol, Performance and Experience
The EpiOcular™ Model Protocol, Performance and Experience Rodger Curren, Ph.D., John Harbell, Ph.D. and Kevin Trouba, Ph.D. Institute for In Vitro Sciences, Inc. EpiOcular™ Outline • Background • Protocol • Performance • Experience EpiOcular™ 1. The EpiOcular™ tissue model is produced by The MatTek Corporation, located in Ashland, Massachusetts. 2. The model estimates the potential ocular irritation of a test article by measuring cytotoxicity (MTT dye conversion) by the EpiOcular™ tissue construct after topical exposure to the test article. 3. The endpoint of the assay, the ET50 value, is the time of exposure to test article required to reduce cell viability to 50% of negative control levels. 4. The EpiOcular™ assay assumes that in vitro cytotoxicity is directly related to in vivo damage that a toxicant would inflict upon an exposed cornea. 5. This is based in part on the hypothesis by Maurer and Jester1 that the level of ocular irritation is related to the extent of initial injury, and that regardless of the processes leading to tissue damage, the extent of initial injury is the principal factor determining the outcome of ocular irritation. 1Extent of initial corneal injury as the mechanistic basis for ocular irritation: key findings and recommendations for the development of alternative assays. Regul Toxicol Pharmacol. 2002 Aug;36(1):106-17. EpiOcular™ Its Position in a Continuum of Sensitivity Rabbit EpiOcular BCOP Extreme Severe Moderate Mild Household Very Mild Color Cosmetics Personal Care Industrial Chemicals EpiOcular™ Cornea http://medicalimages.allrefer.com/large/cornea.jpg http://www.drmingwang.com/Publications/book/22.jpg EpiOcular™ Histology Upper squamous cells Central squamous cells Lower rounded cells NOTE: The EpiOcular™ tissue construct models only the top epithelial layer and not the stromal and endothelial layers of the cornea. 3 EpiOcular™ Tissue Culture Technique: Air/Liquid Interface Tissue Culture Well Culture Insert Tissue Membrane Medium EpiOcularTM Reagents & Equipment EpiOcularTM Basic Procedure 1 min – 24 hr Dose & Incubate 3 hr MTT Addition Reduction 2 hr Quantification Extraction Reagents and Initial Tissue Preparation EpiOcularTM tissues and reagents are shipped overnight cold, and all materials are stored at 4°C. Tissues can be used within 48 hours of receipt. The tissues are examined for obvious defects and may be rejected based on blistering, excess fluid, air bubbles below the tissue insert, etc.. Prior to test article dosing, tissues are transferred (using sterile technique) to 6-well plates that contain fresh assay medium. The tissues are incubated at standard conditions (5% CO2, 37°C, 95% humidity) for at least 1 hour. Reagents and Initial Tissue Preparation Unused EpiOcularTM tissues should be equilibrated with 5% CO2 and stored at 4°C. We perform this step by gassing with 5% CO2 in a storage bag. Our experience indicates that normal equilibration at 5% CO2, 37°C, 95% humidity (i.e., in a tissue culture incubator) may produce variability in tissue performance. Prior to dosing with TA or controls, the tissues are refed with fresh, prewarmed assay medium and generally dosed within 30 minutes of refeeding. Dosing Dosing of aqueous or semi-viscous test articles (TA) is performed with a positive displacement pipette. Solid materials are “sprinkled” onto the surface of the tissue (millicell removed temporarily from well). A dosing device (e.g. the flat end of a sterile push pin) can be used to ensure that the TA covers the complete tissue surface. The tissues are incubated at standard conditions for a specified amount of time. Dosing times generally range from 1 minute to 24 hours. Dosing Device Rinse and Soak The tissues are removed from the incubator and the TA is removed using phosphate buffered saline (PBS). The PBS is sprayed against the millicell wall (not directly on the tissue) to create a gentle vortex which aids in TA removal. The tissues are “soaked” in medium at room temperature to ensure a more complete removal of any remaining TA. Following the soak process, the tissues are rinsed again with PBS prior to the MTT reduction step. Complete TA removal is necessary to prevent prolonged exposure and overestimated toxicity. MTT Reduction and Extraction Individual tissues are placed into wells containing unreduced 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution. The tissues are incubated at standard conditions for 3 hours. A dark blue or purple color signifies the presence of reduced MTT/viable tissue. Following incubation, the tissues are removed and placed into isopropanol for 2 hours with shaking at room temperature to extract the reduced MTT. Transfer of MTT/Isopropanol and Quantification Extracted MTT is thoroughly mixed and transferred to a 96-well plate. The 96-well plate/MTT-isopropanol samples are quantified using a microplate reader. Raw OD550 values are used to to calculate the final ET50 values. 0.3% TRITON®-X-100 Percent of Control 100& Interpretation Graph 80 60 40 Data 20 0 0 10 20 30 40 50 60 Exposure Time (minutes) EpiOcular™ Reproducibility Model Inception = 1996 Positive control: 0.3% Triton-X-100 (Historical Control Range = 27 minutes +/- 2 std. dev.) Negative control: Sterile DI H20 Test Material Mean ET50 Value (min) Standard Deviation (min) CV 26.1 5.4 20.7% 27.0 6.0 22.2% 0.3% Triton X-100 (Combined data from MatTek and IIVS) 0.3% Triton X-100 (IIVS only - through Oct., 2004) SD RAN G E 1997 1998 1999 2000 2001 2002 2003 2004 2005 0.0 0.5 1.0 0.0 1.5 41% 35% 20% 95% 5% 52% 26% 17% 95% 5% 36% 31% 24% 92% 8% 29% 25% 27% 81% 18% 35% 36% 20% 91% 9% 32% 22% 31% 85% 15% 36% 26% 25% 87% 13% 33% 27% 19% 79% 21% 47% 35% 15% 97% 3% 1997-2005 YTD 38% 29% 22% 89% 11% >50 >50 >50 >50 >50 >50 >50 >50 >50 >500 22.9 25.0 22.1 20.7 22.9 22.5 24.1 22.2 24.77 23.00 T O 0.5 T O 1.0 T O 1.5 T O 1.5 T O 2.0 # Production Lots AVE ET 50 (m in) Correlation of In Vitro and In Vivo Results Draize - EpiOcular™ Draize Score1 Classification Example EpiOcular ET50 (min)2 0-15 Non-irritating, Minimal PEG-75 Lanolin > 256-26.5 15.1-25 Mild 3% SDS 26.5-11.7 25.1-50 Moderate 5% Triton X-100 11.7-3.45 50.1-110 Severe, Extreme 5% BAK < 3.45 Based on 20% dilutions for materials with density > 0.95 1Kay, J.H. and Calandra, J.C., Interpretation of eye irritation tests, J. Soc. Cosmetic Chem., 13, 281-289 (1962) 2MatTek Assessment of Ocular Irritation Ranges of Market-Leading Cosmetic and Personal-Care Products Using an In Vitro Tissue Equivalent N E McCain, R R Binetti, S D Gettings, and B C Jones Avon Products, Inc., Suffern, NY, USA Formulation Category Surfactant Formulas Adult Shampoo Children’s Shampoo Baby Shampoo Baby Wash Eye Area Cosmetics Liquid Eyeliner Eye Cream Mascara Pencil Eyeliner Creams/Lotions Adult Lotion/Cream Sunscreen Baby Lotion Non-Surfactant Haircare Children’s Styling Gel Adult Conditioner Children’s Conditioner Children’s Hair Detangler Number of Samples Mean ET50 (min) Range of ET50 Scores (min) 9 7 4 4 27.0 58.2 74.9 114.5 17.1-35.1 37.9-135.4 31.9-107.1 76.9-155.9 15 11 20 3 532.2 581.8 655.1 1440 64-1440 102-1200 199-1080 1440 17 15 2 511.4 673.6 1200 234-1440 252-1440 1200 2 5 2 3 172.7 279.6 728.0 868.4 126-219.3 100-530 256-1200 205.1-1200 10% 100% Test Article and Other Effects Hydroscopic Test Article Direct MTT Reduction Air Bubble 0% Tissue Kill ~ 25% Tissue Kill ~ 60% Tissue Kill Test Article and Other Effects Robust tissue peripherally Residual Test Article Inconsistent MTT reduction- air bubble or test article artifact? EpiOcularTM MTT Reduction Throughout the Tissue (Hines et al, 2003) Verification that MTT reduction occurs in all layers of the EpiOcular™ tissue construct and reasonably reflects cytotoxicity. EpiOcularTM tissue was treated for the indicted time with 0.3% Triton X-100. MTT reduction is dependent on the presence of glucose in the MTT medium. EpiOcular™ Summary & Acknowledgments • • • • Background Protocol Performance Experience The IIVS lab team