Version : 1.2 Page 1 of 16 September 2005 EPIDERMIS MODEL EPISKIN -ECVAM Skin Irritation Validation Study- VALIDATION OF THE EPISKIN SKIN IRRITATION TEST - 42 HOURS ASSAY FOR THE PREDICTION OF ACUTE SKIN IRRITATION OF CHEMICALS. - 42 HOURS EPISKIN SKIN IRRITATION TEST DETERMINATION OF IL-1 α CONCENTRATION IN THE CULTURE MEDIUM S.O.P DRAFT 1
Version : 1.2 Page 2 of 16 September 2005 EPIDERMIS MODEL EPISKIN CONTENTS 1 MATERIALS P 4 1.1 Materials provided with the kit P4 1.2 Materials not provided with the kit P6 2- METHODS P7 2.1 Preparation of samples P7 2.2 Preparation of ELISA reagents and working solutions P7 3 ASSAY PROCEDURE P9 4 DATA REPORT AND CALCULATIONS P12 Annex 1 and 2 : PLATE SCHEDULE P14 2
Version : 1.2 Page 3 of 16 September 2005 EPIDERMIS MODEL EPISKIN - 42 HOURS EPISKIN SKIN IRRITATION TEST Measurement of IL1 α by a Quantitative Sandwich Enzyme Immunoassay Technique (ELISA) IL-1α ELISA kits PRINCIPLE OF THE ASSAY Quantitative sandwich enzyme immunoassay technique. Monoclonal specific IL-1α antibodies are pre-coated onto micro-plates. Standards or samples are added to the wells enabling IL-1α to binding to immobilized antibody. After washing, an enzyme linked polyclonal antibody specific to IL-1α is added to the wells. Unbound antibody was then removed. A substrate solution is added to the wells and a color develops in proportion to the amount of IL-1α bound in the initial step. The intensity of the color (related to the IL-1α amount) is then measured. 1. MATERIALS KIT REFERENCE : Quantikine Human IL-1 a Immunoassay Catalog number DLA 50 (1 plate 96 wells) or SLA 50 (6 plates 96 wells) R&D Systems 3
Version : 1.2 Page 4 of 16 September 2005 EPIDERMIS MODEL EPISKIN 1.1 MATERIALS PROVIDED WITH THE KIT DESCRIPTION IL-1 α Microplate IL-1α Conjugate IL-1α Standard Assay Diluent RD1C Calibrator Diluent RD5 Wash Buffer Concentrate Color Reagent A Color Reagent B Stop solution Plate Covers COMMENT 96 well polystyrene microplate coated with a monoclonal antibody against IL-1α 21ml of polyclonal antibody against IL1α conjugated to horseradish peroxidase 1.25 ng of recombinant human IL-1 α in a buffered protein base with preservatives, lyophilized 6ml of buffered protein base with preservatives. May contain precipitate 21 ml of a buffered protein base with preservatives. For cell culture medium 21 ml of a 25-fold concentrated solution of buffered surfactant with preservatives 12.5 ml of stabilized hydrogen peroxide 12.5 ml of stabilized chromogen 6 ml of 2N sulfuric acid 4 adhesive strips 4
Page 5 of 16 1.2 MATERIALS NOT PROVIDED WITH THE KIT Materials Polypropylene tubes with caps Multi-well plate shaker 96 well plate reader with 450 nm filter Use For media collecting and freezing. For making standard dilution For incubation media homogenizing before collecting For OD readings Wash bottle for collecting fluids For ELISA washing steps Pipettes 5ml For reconstituting the IL-1α standard Adjustable micro-pipette 0 to 200 µl and pipette tips Adjustable micro-pipette 0 to 1000 µl and pipette tips Adjustable multi-step pipette, combitips 2.5 and 5 ml Adjustable multi-channel pipette or plate washer Stop-watches/ timers For media sampling and transfer to ELISA plates For making standard dilution For ELISA reagents For ELISA washing steps For ELISA incubations control Aluminium foil To protect from light Absorbent paper For ELISA washing steps 500 ml of sterile distilled water For diluting wash buffer - 5 -
Page 6 of 16 2. METHODS 2.1 PREPARATION OF SAMPLES Media sampling for mediators and enzyme release measurement after the 42hrs incubation period (see S.O.P EPISKIN SKIN IRRITATION TEST42 hours page18) Tubes Labelling : batches number, chemical code, replicate number, date, 1 tube should be used per epidermis Shake plates containing the treated epidermis (lids on) for 15 minutes, 300 rpm/min. Transfer 1.6ml of homogenized incubation media to pre labeled tubes Incubation Medium from each tissue is stored frozen at -20 C until analyze IL-1 α measurement: thaw frozen incubation media at room temperature and keep at 4 C until use. The time necessary to bring samples to room temperature can vary (depends on room temperature, type of tubes support and density of tubes in the support). Increasing space between tubes helps to accelerate thawing. Do not use warm water bath (cytokine damage). 2.2 PREPARATION OF ELISA REAGENTS AND WORKING STANDARDS (See QUANTIKINE HUMAN IL-1α PROCEDURE, R&D systems) Bring all reagents to room temperature before use - 6 -
Page 7 of 16 Prepare plate schedule Remove excess micro-plates strips from the plate frame (return them to the foil pouch containing the desiccant pack and store at 4 c) Prepare IL-1α standard During thawing time, prepare IL-1α Standard (following notice instructions, page 6). Use Calibrator diluent RD5 to make IL-1 α standard and dilutions. Reconstitute the IL-1α Standard with 5ml of Calibrator Diluent RD5. This reconstitution produces a stock solution of 250. Allow the standard to sit for a minimum of 15 minutes with gentle shaking prior to making dilutions. Pipette 500µl of Calibrator Diluent RD5 into each tube. Use the stock solution to produce a dilution series. Mix each tube thoroughly before transfer. The undiluted standard serves as high standard (250), Calibrator Diluent RD5 as the zero (0). Prepare Positive control dilution (from SDS treated samples) with culture maintenance medium Samples out of standards range should be diluted before testing. For the EpiSkin model the PC should be diluted (1/2). Prepare wash buffer : Dilute 20ml of Wash Buffer Concentrate into sterile distilled water to prepare 500ml of wash buffer. - 7 -
Version : 1.1 September 2005 EPIDERMIS MODEL: EPISKIN Page 8 of 16 3. ASSAY PROCEDURE ¾ ADD 50µl of Assay Diluent RD1C to each well, with an adjustable multi-step pipette (2.5ml combitips) ¾ As reported in the plate schedule: Shake the tube containing samples just before pipetting (one at a time), add 200µl samples or standard to each well, with an adjustable micro-pipette (0-200µl). Cover with the adhesive strip Incubate for 2 hours at room temperature. Single precision pipettes (Eppendorf, Gilson or similar) are used for samples and standards deposit (great precision is necessary). -8-
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Page 10 of 16 Washing step After incubation: Invert the plate to discard the reagents (use a recipient collector) (a) and blot it against clean paper toweling (b). Wash, by filling each well with wash buffer (400µl), using a multi-channel pipette manifold dispenser(c). (a) (b) (c) Repeat washing steps 3 times. After the last wash, dry the plate by gently taping on the paper toweling. Rinsing is an important step. It should be drastic. Drying on paper toweling should be repeated to ensure complete drying and to avoid cross contaminations. Add 200µl of Conjugate to each well, with the adjustable multi-step pipette (5ml combitips). Cover with a new adhesive strip. Incubate 1 hour at room temperature. Repeat the washing step (wash 3 times) Prepare Substrate Solution: Mix extemporaneously color reagents A and B in equal volumes : (use within 15 minutes). Add 200µl Substrate solution to each well, with the multi-step pipette (5ml combitips). - 10 -
Page 11 of 16 Cover with a new adhesive strip protect from light with aluminum foil. Incubate 20 minutes at room temperature. Add 50µl of Stop Solution to each well with the multi-step pipette (2.5ml combitips). If the color does not appear uniform, tape the plate gently. Read at 450nm within 30 minutes. It is not necessary to use the double OD measurement (570 and 450) as recommended in the kit procedure. We used the single 450 nm filter measurement. Standard curve is a linear log/log curve - 11 -
Page 12 of 16 4. DATA REPORT AND CALCULATIONS CORRESPONDENT INFORMATION TO MDS Experiment no. L'oreal phase2 set1 run 1 Tissue lot # 05EKINxxx Date IL1 batch n xxxxx exp.27/09/05 Operator Wavelenght 450 FIXED PLATE DESIGN 1 2 3 4 5 6 7 8 9 10 11 12 A Blank Blank NC NC NC C7 C7 C7 C15 C15 C15 empty B S7 S7 PC PC PC C8 C8 C8 C16 C16 C16 empty C S6 S6 C1 C1 C1 C9 C9 C9 C17 C17 C17 empty D S5 S5 C2 C2 C2 C10 C10 C10 C18 C18 C18 empty E S4 S4 C3 C3 C3 C11 C11 C11 C19 C19 C19 empty F S3 S3 C4 C4 C4 C12 C12 C12 C20 C20 C20 empty G S2 S2 C5 C5 C5 C13 C13 C13 C21 C21 C21 empty H S1 S1 C6 C6 C6 C14 C14 C14 C22 C22 C22 empty standards standards tissue 1 tissue 2 tissue 3 tissue 1 tissue 2 tissue 3 tissue 1 tissue 2 tissue 3 IMPORT 1 2 3 4 5 6 7 8 9 10 11 12 A 0,0518 0,0561 0,0000 B 0,0960 0,0971 0,0000 C 0,1319 0,1349 0,0000 D 0,2008 0,2143 0,0000 E 0,3508 0,3694 0,0000 F 0,6286 0,6246 0,0000 G 1,0765 1,1571 0,0000 H 1,8361 1,9269 0,0000 standards standards tissue 1 tissue 2 tissue 3 tissue 1 tissue 2 tissue 3 tissue 1 tissue 2 tissue 3 Test Chemical N Code N Test Chemical N Code N standards Negative control NC Test Chemical No. 11 11 S1 250 pg/ml Positive control PC Test Chemical No. 12 12 S2 125 pg/ml Test Chemical No. 1 1 Test Chemical No. 13 13 S3 62,5 pg/ml Test Chemical No. 2 2 Test Chemical No. 14 14 S4 31,2 pg/ml Test Chemical No. 3 3 Test Chemical No. 15 15 S5 15,6 pg/ml Test Chemical No. 4 4 Test Chemical No. 16 16 S6 7,8 pg/ml Test Chemical No. 5 5 Test Chemical No. 17 17 S7 3,9 pg/ml Test Chemical No. 6 6 Test Chemical No. 18 18 Test Chemical No. 7 7 Test Chemical No. 19 19 Test Chemical No. 8 8 Test Chemical No. 20 20 Test Chemical No. 9 9 Test Chemical No. 21 21 Test Chemical No. 10 10 Test Chemical No. 22 22-12 -
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Page 14 of 16 5. INTERLEUKIN 1 ALPHA DETERMINATION IL-1α are measured in culture media only for tissues with a viability > 50% after treatment. Mean of IL-1α release concentrations from triplicate tissues was expressed in. For each independent experience, in order to minimize intra and inter-variability, the normalization of the data is completed as follow: Mean [IL-1α ()] = Mean [IL-1α treated tissues] Mean [IL-1α negative control tissues] Whenever the differences of IL-1α release between the test-product and the negative control was negative, the result was set to 0. 6. DATA INTERPRETATION PREDICTIVE MODEL The cut off limit defined for Mean IL-1α is set to 50 The test substance is considered to be irritant (R38) to skin: if the viability after 15 minutes of exposure and 42 hours of post incubation is > 50%, and the amount of Mean Interleukin 1 alpha release is 50 The test substance is considered to be non irritant (no label) to skin: if the viability after 15 minutes of exposure and 42 hours of post incubation is > 50%, and the amount of Mean Interleukin 1 alpha release is < 50. Final Prediction Model combining viability and IL-1α Criteria for in vitro interpretation Mean tissue viability is 50 % Mean tissue viability is > 50 % Classification Irritant (I) R38 Irritant (I) R38 AND Amount of Interleukin 1a released 50 Mean tissue viability is > 50 % Non Irritant (NI) No label AND Amount of Interleukin 1a released < 50 These results are valuable only when using the kits IL-1 alpha R&D systems in reference in this procedure. - 14 -
Page 15 of 16 Annex 1. PLATE SCHEDULE Date: A B C D E F G H Experience: 1 2 3 4 5 6 7 8 9 10 11 12 Diluent RD5 0 3.9 7.8 pg/lm 15.6 31.2 62.5 125 250 Diluent RD5 0 3.9 7.8 pg/lm 15.6 31.2 62.5 125 250 NC 1 Chem.2 3 4 5 6 7 NC 1 2 3 4 5 6 7 NC 1 2 3 4 Chem.5 6 Chem.7 8 9 10 11 12 13 14 15 8 9 10 11 12 13 14 15 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 16 17 18 19 20 21 22 23 16 17 18 19 20 21 22 23 empty empty empty empty empty empty empty empty IL-1 α batch number: Performed by: Signature: - 15 -
Page 16 of 16 Annex 2. PLATE SCHEDULE Date: Experience: 1 2 3 4 5 6 7 8 9 10 11 12 A 0 0 B 3.9 3.9 C 7.8 7.8 D 15.6 15.6 E 31.2 31.2 F 62.5 62.5 G 125 125 H 250 250 IL-1 α standard IL-1 α batch number: Performed by: Signature: - 16 -