Version: 5.0 Draft Standard Operating Procedure page 1 of 31 ...

Version: 5.0 Draft Standard Operating Procedure page 1 of 31

October

2004 IN VITRO SKIN IRRITATION TEST: HUMAN SKIN MODEL

Model: EpiDerm™- 200

Drafted by: Dr. M. Liebsch, ZEBET sign date:

D. Traue, ZEBET sign: date

H. Kandarova, ZEBET sign: date

APPROVAL OF PARTCIPATING LABORATORIES REPRSENTATIVES ZEBET approval Dr. Manfred Liebsch sign: date:

BASF approval

Dr. Armin O. Gamer sign: date:

IIVS approval

Dr. John Harbell sign: date:

NOTE:

This SOP will be used by laboratories participating with the human epidermis model EpiDerm™ in Phase 2 of the ECVAM Skin Irritation Validation Study.

As compared to version 4.0 (used in Phase 1), this version additionally contains the procedure of medium collection and storage of the medium samples for the new IL-1 analysis.

Other changes comprise several improvements in the technical descriptions based on the outcome of the training meeting of the participating laboratories.

In addition, in the process of harmonising the EPISKIN and EpiDerm SOP's used in this validation study, and following recommendations of the ECVAM biostatistician, Assay Acceptance Criterion 3 is now based on the Standard Deviation (SD) between identically treated tissues rather than on the Coefficient of Variation (CV).

Changes (other than simply editorial) are listed below.

Paragraph Changes made in version 5.0 Page

7.3. Test for direct MTT reduction Data interpretation if direct MTT reduction is >30% 8

7.5.1 Assay Acceptance criterion 1 Changed (more rigid) 10

7.5.3 Assay Acceptance criterion 3 Changed from CV to SD 10

7.6.1. MTT solution MTT medium preparation from powder added 11

7.8 Experimental procedure

MTT test and medium collection

procedure for medium collection and storage added

16

ANNEX B - MDS improved 22

ANNEX C - Characterisation of test sub-stances

New, not part of the MDS, but highly recommended to use

30


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CONTENTS page

1 RATIONALE AND BACKGROUND .........................................................................................................3

2 SPECIFIC PURPOSE OF THE METHOD.................................................................................................3

3 BASIS OF THE METOD............................................................................................................................4

3.1 TEST SYSTEM ..........................................................................................................................................4

4 LIMITATIONS OF THE METHOD.............................................................................................................5

5 BRIEF BASIC PROCEDURE....................................................................................................................5

6 DATA INTERPRETATION PROCEDURE................................................................................................5

7 MATERIALS AND METHODS..................................................................................................................6

7.1 MATERIAL PROVIDED BY MATTEK..............................................................................................................6

7.1.1 Epi-200 Kit Components ...................................................................................................................6

7.1.2. MTT-100 ASSAY KIT COMPONENTS .....................................................................................................6

7.1.3 Expiration and Kit Storage ................................................................................................................6

7.2 MATERIALS NOT PROVIDED WITH THE MATTEK KITS ...................................................................................7

7.3 TEST FOR DIRECT MTT REDUCTION ..........................................................................................................8

7.4 TEST FOR MESH COMPATIBILITY (LIQUID TEST SUBSTANCES ONLY).............................................................9

7.5 ASSAY QUALITY CONTROLS....................................................................................................................10

7.5.1 Assay Acceptance Criterion 1: Negative Control .......................................................................10

7.5.2 Assay Acceptance Criterion 2: Positive Control .........................................................................10

7.5.3 Assay Acceptance Criterion 3: Coefficient of variation ..............................................................10

7.6 PREPARATIONS......................................................................................................................................11

7.6.1 MTT solution (prepare freshly on day of testing)........................................................................11

7.6.2 Dulbeccos PBS...........................................................................................................................11

7.6.3 5% (aq) SDS...............................................................................................................................11

7.7 TEST SUBSTANCES ................................................................................................................................12

7.8 EXPERIMENTAL PROCEDURE (SEE ALSO ANNEX A) ................................................................................14

7.9 DOCUMENTATION...................................................................................................................................18

7.9.1 Method Documentation Sheet, MDS (see ANNEX B)................................................................18

7.9.2 Test Data ....................................................................................................................................19

8 REFERENCES ........................................................................................................................................20

ANNEX A: EPIDERMTM SKIN IRRITATION TEST: FLOWCHART..............................................................21

ANNEX B: METHODS DOCUMENTATION SHEET (MDS )........................................................................22

ANNEXC: CHARACTERISATION OF TEST SUBSTANCES......................................................................30


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1 RATIONALE AND BACKGROUND

Dermal irritation is generally defined as “the production of reversible inflammatory changes in the skin” (1). The potential of chemicals to induce skin irritation (hazard) is an important consid-eration in establishing procedures for the safe handling, packing and transport of chemicals. Skin irritation is usually determined by in vivo rabbit skin irritation test (2) as described for example in the OECD TG 404 (1).

Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are suffi-ciently complex to mimic human skin barrier and cell reactivity.

Upon review of existing information by the EVAM Skin Irritation Task Force (3) and an ECVAM Workshop (4) several in vitro systems and toxicological endpoints were regarded as suffi-ciently promising predictors for skin irritancy potential, and an ECVAM prevalidation study was conducted (5) employing in a final blind trial 20 chemicals from the ECETOC Data Base No. 66 “Skin Irritation/ Corrosion” (6). The Study outcome revealed the failure of two tests and the need to refine three tests (two 3D human epidermal model tests and the mouse ex vivo “Skin Integrity Function Test (SIFT)” that had showed a sufficient reproducibility, but unbalanced predictivity (5)(7). As a consequence, the latter three tests had to step back into refinement of the test proce-dures and prediction models (7)(8), first conducted with EPISKIN (9), and later with EpiDerm and SIFT (personal communications of the lead laboratories to the ECVAM Skin Irritation Task Force on 21.11.2002). To facilitate future acceptance of the tests at the OECD level the refinement re-sulted in a common skin model test protocol that will be used by EPISKIN and EpiDerm, and only differs in model specific details.

2 SPECIFIC PURPOSE OF THE METHOD

The In vitro method is designed to predict skin irritation potential of neat test substances in the context of identification and classification of skin irritation hazard according to EU classification system (R 38 or no label), using reconstructed human epidermal model EpiDerm TM.

Because the refinements of the method aimed in a better separation of R 38 irritants from non irri-tants the method cannot be used for prediction of mild irritants according to the Globally Harmo-nised System (GHS) for classification and labelling.

This method has been developed and refined by L’Oreal ((9) and J. Cotovio, personal communica-tion at the ECVAM workshop, May 2003) and later applied on EpiDerm (M. Liebsch, personal communication at the MT meeting 17/18 November 2003, (10) with the aim to develop for both models a common protocol able to predict skin irritation potential according to the EU classifica-tion system and replace the in vivo acute skin irritation test in rabbits.


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3 BASIS OF THE METHOD

The test consists of a topical exposure of the neat test chemical to a human reconstituted epider-mis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide], present in cell mitochon-dria, into a blue formazan salt that is quantitatively measured after extraction from tissues (11). The reduction of viability of tissues exposed to chemicals in comparison to negative controls (treated with water) is used to predict skin irritation potential. Recent comparative studies in human skin models employing various endpoints to predict skin irritancy of topical formulations have shown that the MTT endpoint had clear advantages, even over mechanistically based endpoints like the release of IL-1α (12)(13).

3.1 Test system

Test system description: The EpiDerm™ System (MatTek, Ashland, USA) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. A generic description of general and functional conditions that skin models need to comply with can be found in the new OECD Test Guideline 431 In vitro Skin Corrosion: Human Skin Model (14).

The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELLS, 10 mm ∅) and shipped world-wide as kits, containing 24 tissues on shipping aga-rose together with necessary amount of culture media and 6-well plates. In addition the MTT kit (containing MTT concentrate, diluent, extractant, PBS and 24- well plate) is provided by MatTek.

Quality control: The EpiDerm™ System is manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium are tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination.

MatTek provides also information about ET 50 of Triton X-100 (1%) and tissue viability (MTT test) for each EpiDerm lot, together with historical database of results.

Precautions: The epidermal cells are taken from healthy volunteers negative to HIV, and Hepatitis. Neverthe-less, handling procedures for biological materials should be followed:

a) it is recommended to wear gloves during handling with the skin and kit components

b) after use, the epidermis, the material and all media in contact with it should be decontaminated prior to disposal (e.g. using 10 % bleach or special containers)

Note: Due to long postincubation period, it is necessary to perform the test under aseptic conditions in the microbiologically safety cabinet (laminar flow hood)


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4 LIMITATIONS OF THE METHOD The method has so far not fully been validated. One limitation that became obvious in other studies is a possible interference of the test substance with the endpoint MTT. A test substance may di-rectly reduce MTT and mimic dehydrogenase activity of the cellular mitochondria. This property of the test substance is only a problem, if at the time of the MTT test (here: 42 hours after test sub-stance exposure) still sufficient amounts of the test substance are present on (or in) the tissues. In case of this unlikely event, the (true) metabolic MTT reduction and the (false) direct MTT reduction can be differentiated and quantified by a procedure described in section 7.3. The method is not designed for testing of highly volatile test substances.

5 BRIEF BASIC PROCEDURE

On day of receipt, EpiDerm tissues are kept in the refrigerator. Next day, tissues are conditioned by incubation for release of transport-stress related compounds and debris. After 1 hr of pre-incubation tissues are transferred to fresh assay medium (0.9 mL , 6-well plates) and topically ex-posed to the test chemicals for 15 minutes. Three tissues each are used per test chemical (TC), positive control (PC) and negative control (NC). Tissues are then thoroughly rinsed an blotted to remove the test substances, and transferred to fresh medium. After a 42 hrs incubation period, the MTT assay is performed by transferring the tissues to 24 well plates containing 0.3 mL MTT me-dium (1mg/mL). After a 3 hrs MTT incubation the blue formazan salt formed by cellular mitochon-dria is extracted with 2 mL isopropanol per tissue (24-well plates, 2 hrs, shaker) and the optical density of the extracted formazan (200 µl) is determined in a spectrophotometer at 570 nm. Rela-tive cell viability is calculated for each tissue as % of the mean of the negative control tissues. Skin irritation potential of the test materials is predicted if the remaining relative cell viability is below 50% (paragraph 6)

6 DATA INTERPRETATION PROCEDURE (Prediction Model) Note: According to recent international agreements (OECD Validation Workshop, 2002) any new method used for identification of human health or ecological hazards will need a Data Interpretation Procedure (DIP), defined for the specific purpose or use of a test. In case of in vitro methods used for prediction of in vivo hazards, the DIP is often an empirically or biostatistically based mathematical rule, also called Prediction Model (PM).

For the current test, an irritation potential of test materials according to EU classification (R38 or no label) is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below 50% of the mean viability of the negative controls.

In vitro result In vivo prediction

mean tissue viability ≤ 50 Irritant (I), R38,

mean tissue viability > 50 non-irritant (NI)


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7 MATERIALS AND METHODS

7.1 Material provided by MatTek

7.1.1 Epi-200 Kit Components

Epi-200 kits are shipped from Boston on Monday. If possible, make sure that they are arriving in the laboratory on Tuesday. Upon receipt of the EpiDerm tissues, place the sealed 24 well plates and the assay medium into the refrigerator (4°C). Place the MTT concentrate containing vial in the freezer (-20°C) and the MTT diluent in the refrigerator (4°C).

Record lot numbers of all kit components in the Methods Documentation Sheet (MDS) (see ANNEX B)

1 Sealed 24-well plate (EPI-200) Contains 24 inserts with tissues on agarose

2 24-well plates (sterile) Use for MTT viability assay

4 6-well plates (sterile) Use for assay

1 bottle, 60 ml Assay Medium (EPI-100-ASY) DMEM-based medium

1 vial, 10 ml 1% Triton X-100 Solution (TC-TRI) Skin irritant reference chemical Do not use in present method

7.1.2. MTT-100 Assay Kit Components

1 vial, 2 ml MTT concentrate (MTT-100-CON)

1 vial, 8 ml MTT diluent (MTT-100-DIL) For diluting MTT concentrate prior to use in the MTT assay

1 bottle, 60 ml Extractant Solution (Isopropanol) For extraction of formazan crystals

1 bottle, 125 ml PBS Rinse Solution (TC-PBS) Use for rinsing the inserts in MTT assay

1 MTT Assay Protocol MatTek Corporation: steps are in-cluded in the present protocol

7.1.3 Expiration and Kit Storage

part # description conditions shelf life

EPI-200 EpiDerm cultures refrigerator (4°C) 96 hours

EPI-100-ASY Assay medium refrigerator (4°C) 7 days

MTT-100-DIL MTT diluent refrigerator (4°C) 2 months

MTT-100-CON MTT concentrate freezer (-20°C) 2 months

Note : examine all kit components for integrity. If there is a concern call MatTek Corporation immediately (Mitch Klaus-ner, +1-508-881-6771, Email: [email protected]).

mailto:[email protected])


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7.2 Materials not provided with the MatTek kits

• Laminar flow hood For safe work under sterile conditions

• humidified incubator (37°C, 5% CO2, 95 % relative humidity)

For incubating tissues prior to and during assays

• Vacuum source/trap (optional) For aspirating media and solutions

• Laboratory balance For pipette verification and checking spoonful weight

• 96-well plate photometer For reading OD

• Plate shaker For extraction of formazan

• Stop-watches To be used during application of test materials

• Sterile, blunt-edged forceps For handling tissue inserts

• 500 mL plastic wash bottle For rinsing tissue with PBS

• 200 mL beaker For collecting PBS rinses

• 37°C water bath For warming Media and MTT solution

• Mortar and Pestle For grinding granulars

• Adjustable Pipette / multi-step Pipette For pipetting 0.9 mL assay medium

• Adjustable Pipette / multi-step Pipette For pipetting 300 µL MTT medium

• Adjustable Pipette For pipetting 2 mL MTT extraction solu-tion

• Adjustable Pipette For pipetting 200 µL formazan extract from 24-well plate into 96-well plate for the plate photometer

• Adjustable Pipette For application of 25 µL liquid test mate-rials

• Positive displacement pipet 25 µL For application of semi-solid test materi-als

• sharp spoon (NaCl weight: 25 mg) For application of solids

Aesculap, Purchase No.: FK 623

• (bulb headed) sound To aid levelling the spoon (spoonful) and spreading of test substances

Dulbeccos PBS without Ca2+ and Mg2+ Biochrom # L 1825 or PAN Biotech # P04-36 500

Use for rinsing tissues.

• Mesh (Nylon 150 µm, ref: 03150/44)

Use as a spreading aid for liquid test materials,

BUISINE, 15 rue de Saint-Juste 60600 Clermont-de-L´Oise, France Email: [email protected]

mailto:[email protected]


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• Biopsy punch d = 8 mm

For the mesh forming

For example: STIEFEL laboratorium Mühlheimer Straße 231 63075 Offenbach am Main, Germany

• MTT - Thiazolyl Blue Tetrazolium

Bromide (Sigma, # M-5655, cell

culture tested, purity min. 97,5 %)

For the MTT assay in case that MTT kit provided by MatTek is not used

• 5 % (aq) SDS [151-21-3]

(Sigma # L-4509, purity min. 98.5%)

To be used as positive control with each kit

• Sterile H2O (distilled or aqua pure) To be used as negative control with each kit

• Extra 6-well plates - sterile (FALCON recommended)

To transfer tissue inserts to fresh media (instead of replacing the media using the same plate)

• Cryovials - polypropylene

(NeoLab # 7-4581)

Collecting and freezing of media samples for each tissue

• Adhesive tape

(NeoLab # 7-2220 or # 2-5082)

Covering plates during formazan extrac-tion

• Cotton tip swabs (sterile) For drying the tissue surface

7.3 Test for direct MTT reduction As specified in paragraph 4, a test substance may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present in the tissues when the MTT viability test is performed. To identify this possible interference, each test substance is checked for its ability to reduce MTT directly (Step 1). In case the test material has a potential to directly reduce MTT, the MTT assay is performed in parallel on viable and on freeze killed tissues (functional check, Step 2).

Step 1:

Add 25 µL (liquid) or 25 mg (solid - using sharp spoon 7.2) of the test substance to 1 mL of the MTT medium and incubate in the dark at 37° C (incubator, 5% CO2) for 60 min. Untreated MTT medium is used as control.

Figure1 : Example of tests for direct MTT reduction ability (Step 1). Test substances (2) (3) and (6) have directly reduced MTT.


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If the MTT solution turns blue/purple, the test substance is presumed to have reduced the MTT, and a functional check should be performed (Step 2).

Step 2: Employs freeze killed tissues that possess no metabolic activity but absorb and bind the test sub-stance like viable tissues.

• Freeze killed tissues are prepared by placing EpiDerm tissues in a freezer (-18°C) for 24 h. Once killed, the tissues may be stored indefinitely in the freezer.

• Each MTT reducing chemical is applied on two freeze-killed tissue. In addition to that, two freeze killed tissues remains untreated. (The untreated killed controls shows small amount of MTT reduction due to residual reducing enzymes within the killed tissue). Data are then cor-rected as follows:

Data correction procedure

True viability = Viability of treated tissue – MTT conversion by chemical = OD tvt – OD kt

OD kt = (mean OD tkt – mean OD ukt)

tvt = treated viable tissue kt = killed tissues

tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)

Interpretation: If the direct reduction by the test substance is greater than 30% of the negative control value, addi-tional steps must be taken into account or the test substance may be considered incompatible with this test system (expert judgement).

If the direct reduction of MTT by the test substance is less than 30 % of the negative control value, the net OD of the test substance treated killed control may be subtracted from the mean OD of the test substance treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only

Note : If the MTT reducing chemical is in the test classified as irritant (tissue viability below 50 %), MTT cor-rection procedure is necessary.

7.4 Test for Mesh Compatibility (liquid test substances only) Capillary effects (surface tension effects) were observed if a low volumes of liquid test chemicals were applied on EpiDerm surface. Therefore, a nylon mesh (see 7.2) is used as a spreading sup-port (see 7.7)

Figure 2: Nylon mesh

The supplier doesn't provide the mesh in circular shape. Therefore, it is necessary to prepare it in the laboratory using the biopsy punch (d = 8 mm) see 7.2.

Note: sterilisation can be done by UV-rays. Autoclaving of nylon is not recommended.


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The nylon mesh can be used for wide range of liquid substances. However, some chemicals may react with the mesh and therefore the compatibility of each liquid chemical with nylon mesh has to be checked.

To test if a test chemical interacts with the mesh, place the mesh on a slide and apply 25 µL test substance. After 15 minutes exposure, check using a microscope (Figure3), if an interaction be-tween test substance and the mesh is noticed (picture b). In that case the test substance has to be applied without using a mesh as a spreading aid.

Figure 3 : The mesh compatibility test

7.5 Assay Quality Controls

7.5.1 Assay Acceptance Criterion 1: Negative Control

The absolute OD of the negative control (NC) tissues (treated with H2O) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.

The assay is meeting the acceptance criterion if the mean OD570 of the NC tissues is ≥ 1.0.

7.5.2 Assay Acceptance Criterion 2: Positive Control A 5 % aq SDS (in H2O) solution see 7.6.3 is used as positive control (PC) and tested con-currently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day.

Viability of positive control should be within 95% confidence interval of the historical data which is in case of EpiDerm (11,6 % - 14,7% ).

The assay is meeting the acceptance criterion if the mean viability of PC expressed as % of the negative control ≤ 20%.

7.5.3 Assay Acceptance Criterion 3: Standard deviation Since in each test skin irritancy potential is predicted from the mean viability deter-mined on 3 single tissues, the variability of tissue replicates should be acceptably low.

The assay is meeting the acceptance criterion if the SD calculated from individual per-centual tissue viabilities of the 3 identically treated replicates is < 11


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7.6 Preparations

7.6.1 MTT solution (prepare freshly on day of testing)

Thaw the MTT concentrate (MTT-100-CON) and dilute with the MTT diluent (MTT-100-DIL). Store the remaining MTT solution in the dark at 4°C for later use on the same day (do not store until next day since MTT will degrade with time).

Safety precaution: MTT (R26, R68, R22, R36, R37, R38) is toxic. Wear protective gloves during manipulation with MTT solution.

Note: MTT is light sensitive. Protect all solutions from light

If you are not using the MTT-kit provided by MatTek, prepare first the stock solution (5mg/ml) of MTT (see 7.2) in PBS. Stock solution can be stored frozen (-20 °C) up to one month.

Before use, filter the stock solution and dilute the filtrate with the assay medium to final concentration (1mg/ml). Record the preparation in the MDS. Do not store this solution longer than 2 hours.

7.6.2 Dulbeccos PBS

Sterile ready-to-use PBS can be used (cat. no. see 7.2). About two litres are sufficient for all rinsing performed with one kit. If PBS is prepared from 10x concentrates or powder pH needs to be adjusted and solution should be sterilised. Record the preparation in the MDS.

7.6.3 5% (aq) SDS

Weigh out 0,50 g SDS (analytical grade, see 7.2) into an appropriate calibrated 10 ml flask (narrow neck) and complete with distilled water to 10 ml (final volume). For better solubili-sation of SDS place solution in a water bath (37°C) for 10 minutes. Solution can be stored up to one month (2 - 8 °C). Record the preparation in the MDS.


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7.7 Test substances

Safety Instruction 1. For handling of non-coded test substances follow instructions given in the Material Safety Data Sheet. 2. If coded chemicals are supplied, no (or possibly incomplete) information regarding the safe handling will

be provided. Therefore, all test materials must be treated as if they were irritating and toxic and work must be performed in accordance with chemical safety guidelines (use ventilated cabinet, wear gloves, protect eyes and face).

3. Store all test substances according to recommendations. Respect special store conditions (special tem-perature, protection from light, protection from oxidisation by nitrogen, etc.)

Liquids: Dispense 25 µL directly atop the tissue and gently place nylon mesh - diameter 8

mm (see 7.2 and 7.4) on the surface. Record the use of mesh as spreading tool in the MDS.

Figure 4: Application of liquids

Semisolids: Dispense 25 µL using a positive displacement pipette directly atop the tissue. If necessary spread to match size of tissue.

Record the use of spreading in the MDS.

Figure 5: Application of semisolids (positive displacement pipette - detail)

Solids: Crush and grind test material in a mortar with pestle wherever this improves the consistency. Fill 25 mg sharp application spoon (see 7.2) with fine ground test ma-terial. Level the spoon by gently scratching the excess material away with an ap-propriate aid, avoiding compression ("packing") of the test material. "Packing" can be avoided by using a rod shaped sound instead of a flat spatula. If a bulb headed sound is used the bulb can be used to empty the spoon completely. Add 25 µL H20 for wetting of the test material to increase tissue surface contact. Increase vol-


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ume of H20 in case of materials where this is not enough for wetting. If necessary spread to match size.

Record in the MDS if grinding was not used and if spreading or increasing of H20 volume was necessary. After application of water to the solid test substance, re-cord observations of the physical description in the MDS.

Figure 6: Application of solids

waxes: For test substances with waxy consistence the spoon application does not work. In these cases try to form a flat “cookie like” piece of about 8 mm diameter and place it atop the tissue, wetted with sterile H2O. To improve the contact between test substance and tissue weigh down the “cookie” with a stainless steel aid like that shown in Figure 7.

The stainless steel aid will be provided to participating labs by the lead-lab.

Figure 7:

Note: Even though test substance exposure time in this test is only 15 minutes, and exposure takes place outside the incubator, highly volatile toxic test substances may affect neighboured tissues within the same 6-well treatment plate. In these cases plates should be covered with an adhesive plate cover, or other meas-ures should be taken into account, like testing the volatile substances on separate plates.


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7.8 Experimental Procedure (see also ANNEX A)

Day prior to testing (day 0) • Upon receipt of the EpiDerm kit(s), place the sealed 24 well plates containing the tissues and

the assay medium into the refrigerator (5 ± 2° C). Place the vial containing the MTT concen-trate in the freezer (-20°C).

• If necessary, prepare sufficient amount of rinsing PBS for the next day.

Chemical exposure (day 1) Tissue conditioning

• Pre-warm the assay medium to 37°C , e.g. in a water-bath.

• Pipette 0.9 mL of the pre-warmed assay medium into each well of sterile 6-well plates.

• Remove the shipped multiwell plate from the refrigerator. Under sterile conditions open the plastic bag containing the 24-well plate with epidermal tissues. Under a sterile airflow remove the sterile gauze and carefully (using sterile forceps) take out each insert containing the epi-dermal tissue. Remove any remaining agarose that adheres to the outer sides of the insert by gentle blotting on the sterile filter paper, and place the tissues in the empty, sterile 24-wellplate and dry the surface of the tissues with a sterile cotton tip swab. Evaluate visually tissue sur-face under a dissecting stereoscope, record observations in the MDS, and transfer tissues to a 6-well plate with 0.9 mL medium.

•

Note (1): any air bubbles trapped underneath the insert should be released.

Note (2): The visual quality check of the tissues has to be done quickly

Note (2): for each chemical use one 6-well plate and place three inserts in the upper row of the pre-pared 6-well plate. Use this design also for the negative and positive control.

• Place the 6-well plates containing the tissues into a humidified (37°C, 5 % CO2) incubator for 1 hour.

• After 1 hour pre-incubation, transfer each insert to fresh medium in the lower row of the six-well plate as shown in Figure 8:

Figure 8: Pre-incubation plate design

• Before test substance exposure, label the 6 well plate lids with the test material codes.


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Test Substance Exposure

• Apply 25 µL or 25 mg of the undiluted test substance, NC or PC to three single tissues each according to 7.7. Dose tissues at the time intervals needed later for rising off the test sub-stances (1 minute). Note: If a single technician is dosing and rinsing tissues, do not dose more than 5 substances (=15 tis-sues) in a block, so to be ready to wash off the test substance after 15 minutes from the first dosed tis-sue.

• Keep dosed tissues in the laminar hood at room temperature (18 – 21°C).

• After 15 ± 0,5 min test substance exposure rinse tissues with sterile PBS, filling and emptying the tissue insert 10 times to remove any residual test material, (Figure 9).

Than 3 times completely submerge insert in 150 ml PBS (gentle shake to remove all rests of test material). Finally, rinse the tissues once with sterile PBS from the washing bottle.

Note (1): Make sure that the nylon mesh is removed from the tissue surface during the first phase of washing procedure (10 times washing). If the mesh is still present on the surface, take it carefully out with forceps and continue with the second step of the washing procedure. Note (2): If the test substance reacts with the insert, the mesh may stick to the edge of insert.

Figure 9: fill with PBS & empty PBS (10 times) • Remove excess PBS by gentle shaking the insert, blot insert bottom on sterile blotting paper

(Figure 10). Transfer blotted tissue inserts to new 6 well plates pre-filled with fresh assay me-dium. Carefully dry the surface of each tissue with a sterile cotton tipped swab (Figure 11). Evaluate visually tissue surface under a dissecting stereoscope. In case that rest of the chemi-cal is still present on the surface, try to remove it with the sterile wetted cotton swab. Record this procedure in the MDS.

Figure 10: blotting Figure 11: drying of the surface

• Incubate tissues at in an incubator for 42 hours. Record start time of incubation in the MDS.

Note (1): The 6-well plates for 42 hours post-incubation should be prepared and labelled. Mark both bottoms (plates) and lids that belong together.


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MTT Test and medium collection (day 3) Medium collection procedure for IL-1 analysis

• Before the 42 hrs incubation is completed, label an appropriate amount of polypropylene cryo tubes with caps (one per tissue ) with:

- chemicals code

- replicate number

- number or run and date.

• At the end of the 42 hours +/- 2 hours incubation period, place the plates on plate shaker for 10 minutes +/- 2 min, speed (500 rpm/min). This step helps to homogenise the released media-tors in the medium before sampling.

• Transfer 0,7 ml of medium from each well to the pre labelled cryo-tubes (see 7.2). Store the tubes in the freezer at - 20°C until analysis. Samples can be stored 6 months.

MTT Test

• Prior the MTT assay, label sufficient amount of 24-well plates for test.

• Prepare MTT medium from frozen concentrate according to 7.6.1 and pipette 300 µL of MTT medium in each well.

• After the medium collection procedure is completed, remove inserts from the 6-well plates, blot bottoms, and transfer them into the 24-well plates, prefilled with 0,3 ml of MTT (1 mg/ml). Place these in the incubator (37°C, 5% CO2), record start time of MTT incubation in the MDS and incubate for 3 hours.

Note: The 3 hours +/- 5 min MTT incubation time must be strictly adhered to. Deviations from the 3 hour time for MTT incubation will result in different MTT readings.

• After MTT incubation is completed, gently aspirate MTT medium from all wells (e.g. using a suction pump plus toxic waste trap). Refill wells with PBS and aspirate again. Repeat this rins-ing twice and make sure that tissues are dry after the last aspiration. Transfer inserts to new 24-well plates.

• Immerse the inserts by gently pipetting 2 mL extractant solution (isopropanol) into each insert. The level will rise above the upper edges of the insert, thus completely covering the tissues from both sides.

• Seal the 24 well plates (e.g. parafilm or gas non-permeable adhesive tape) to inhibit isopropa-nol evaporation. Record start time of extraction in the MDS and extract formazan for at least 2 hours at room temperature with gently shaking on a plate shaker (~ 120 rpm).

As an alternative, overnight extraction is also possible. Seal plates as described above and extracted at room temperature in the dark, without shaking. Before using the extracts, shake for at least 15 min on plate shaker.After the extraction period is complete, pierce the inserts with an injection needle (~gauge 20, ~ 0.9 mm diameter) and allow the extract to run into the well from which the insert was taken. Afterwards the insert can be discarded. Before transferring the extract to 96 well plates pipette up and down 3 x until solution is homogenous.


October



Figure 11: Piercing of the tissue

• Per each tissue transfer 2 × 200µL aliquots of the blue formazan solution into a 96-well flat bot-tom microtiter plate according to fixed plate design given in spreadsheet (example is given in Figure 12). Use isopropanol as blanks. Read OD in a 96-well plate spectrophotometer using a wavelength between 540 and 595 nm, preferably at 570 nm, without using a reference filter (see Figure 13).

PLATE 1

1 2 3 4 5 6 7 8 9 10 11 12 BL BL BL BL BL BL A

Tissue 1 NC PC C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 B

NC PC C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C

Tissue 2 NC PC C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 D

NC PC C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 E

Tissue 3 NC PC C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 F

NC PC C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 G

H

NC - Negative control ; PC – Positive control; C1, C2... - Chemical No. 1, Chemical No.2..; BL - Blank

Figure 12.: Fixed 96 well-plate design (for OD reading in plate photometer, 2 aliquots per tissue)

Note : In contrast to normal photometers, in plate readers pipetting errors influence the OD. Therefore, 2 formazan extract aliquots shall be taken from each tissue extract. In the Excel data sheet these 2 ali-quots will be automatically reduced to one value by calculating the mean of the two aliquots. Thus, for calculations from each single tissue only one single mean OD-value is used.

Note: the plate design is dictated by the plate design used in the Spreadsheet, which is used in the validation study for data collection and preliminary calculations. It is necessary to strictly keep the plate design given here. Otherwise, the calculation of results will be incorrect.


October



Figure 13: Formazan spectrum in isopropanol

Note: Readings are performed without reference filter, since the "classical" reference filter often used in the MTT test (630 nm) is still within the absorption curve of formazan. Since filters may have a ± tolerance in some cases the reference filter reduces the dynamics of the signal (OD) up to 30%.

7.9 Documentation

7.9.1 Method Documentation Sheet, MDS (see ANNEX B)

In addition to PC raw data, the MDS allows a GLP compliant QC of the correct set up, calibration and function of the equipment, as well as all preparations.

If the test is performed as a GLP compliant procedure in a non-GLP environment filling in all infor-mation is mandatory. However, in a full GLP certified laboratory many records (in particular equipment calibrations, temperatures etc.) may be recorded centrally by other means. In this case, reference to e.g. the relevant laboratory notebook is sufficient. Per each experiment, make a hardcopy of the MDS, fill in and sign the requested information, start-ing the day prior to testing and ending after the test has been conducted.

Note (1): If several tests are performed per week, pipette verification (weighing H20 on a balance) is only necessary once at the beginning of each week. If adjustable pipettes are used the correct adjustment shall be checked and recorded in the MDS on each test day.

Note (2): It is recommended to check the weight of a levelled application spoon of each solid test substance and record this weight in the Test Substance Characterization form.


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7.9.2 Test Data

A blank MS EXCEL spreadsheet EpiDerm-SIT-SPREAD.XLS is provided by ZEBET and a copy should be made before the first data entry. The spreadsheet consists of three single maps named: IMPORT, SPREAD and REMARKS.

Data files of optical densities (ODs) generated by the microplate reader (without blank substrac-tion) are copied from the reader software to the Windows Clipboard and then pasted into the first map of the EXCEL spreadsheet (IMPORT). The blank corrections, calculation of results and statis-tical parameters are done automatically in the second part of the spreadsheet (SPREAD).

Use the fixed 96-well plate design as is specified in the SOP (see 7.8).

In addition to the PC entry of the reader raw data, some requested information has to be filled in the first map of the spreadsheet: (tissue lot numbers, test material codes, date, lab personal).

After data entry the spreadsheet performs on a second map the following calculations

1. Blank correction

2. For each individual tissue treated with a test substance (TS), the positive control (PC) and the negative control (NC) the individual relative tissue viability is calculated according to the follow-ing formulas

Relative viability TS (%) = [ODTS / Mean of ODNC] x 100 Relative viability NC (%) = [ODNC / mean of ODNC] x 100 Relative viability PC (%) = [ODPC / mean of ODNC] x 100

3. For each test substance, negative control and the positive control the mean relative viability of

the three individual tissues is calculated and used for classification according to the Prediction Model (see section 6).

4. The spreadsheet shows a graph of the results (% of relative viability ± SD).

• Per each experiment, make a hardcopy of the raw data (i.e. outcome of the reader data).

• Per each experiment, save your secondary data in one copy "EpiDerm-SIT-SPREAD.XLS".

• Fill in the requested information in "EpiDerm-SIT-SPREAD.XLS".

• In addition, per each experiment, keep signed hardcopies of "EpiDerm-SIT-SPREAD.XLS" together with the signed hardcopy of the MDS.


October



8 REFERENCES 1. OECD, 2002. OECD Guideline for Testing of Chemicals, No. 404: Acute Dermal Irritation/Corrosion.

Organisation for Economic Cooperation and Development, Paris

2. Draize J.H., Woodand G.& Calvery H.O. 1944. Methods for the study of irritation and toxicity of sub-stances applied topically to the skin and mucous membranes.Journal of Pharmacology and Experimen-tal Therapeutics. 82, pp 337-390

3. Botham, P.A., Earl, L.K., Fentem, J.H., Roquet, R., Van DE Sandt, J.J.M., 1998. Alternative methods for skin irritation testing: the current status, ECVAM Skin Irritation Task Force Report 1. ATLA 26, 195-211.

4. Van de Sandt, J., Roguet, R., Cohen, C., Esdaile, D., Ponec, M., Corsini, E., Barker, C., Fusenig, N., Liebsch, M., Benford, D., de Brugerolle deFraissinette, A., Fartasch, M., 1999 The Use of Human Keratinocytes and Human Skin Models for Predicting Skin Irritation. The Report and Recommendations of ECVAM Workshop 38. ATLA 27, 723-743

5. Fentem, J.H., Briggs, D., Chesné, C., Elliott, G.R., Harbell, J.W., Heylings, J.R., Portes, P., Roguet, R., Van De Sandt, J.J.M., Botham, P.A., 2001 A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicoloy in Vitro 15, 57-93

6. ECETOC, 1995. Skin Irritation and Corrosion: Reference Chemicals Data Bank ECETOC Technical Report No. 66. European Centre for Ecotoxicology and Toxocology of Chemicals, Brussels

7. Robinson, M.K., Cohen, C., de Brugerolle de Fraissinette, A., Ponec, M., Whittle, E., Fentem, J.H., 2002 Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingre-dients and finished products. Food and Chemical Toxicology 40, 573-592

8. Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliott, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., Van De Sandt, J.J.M., Wiemann, C., Worth, A.P., 2002 Follow up to the ECVAM prevalidation study on in vitro tests for acute skin irritation, ECVAM Skin Irritation Task Gorce Report 2. ATLA 30, 109-129

9. Portes, P., Grandidier, M.-H., Cohen, C., Roguet, R., 2002 Refinement of the Episkin protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicol-ogy in Vitro 16, 765-770

10. Kanďárová H., Liebsch M., Genschow E., Ingrid Gerner, Traue D., Slawik B., Spielmann H. 2004: Op-timisation of the EpiDerm Test Protocol for the upcoming ECVAM Validation Study on In Vitro Skin Irri-tation Tests. ALTEX 21, 3/04, 107-114

11. Faller, C., Bracher, N., Dami, N., Roguet 2002 Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in Vitro 16 (5), 557-572

12. Faller, C., Bracher, M., 2002. Reconstructed skin kits: Reproducibility of cutaneous irritating testing. Skin Pharmacol Appl Skin Physiol 15 (suppl 1), 74-91

13. Mossman, T., 1983. Rapid colorimetric assay for cell growth and survival application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63

14. OECD, 2003. OECD Guideline for Testing of Chemicals, No. 431:In Vitro Skin Corrosion: Human Skin Model Test. Organisation for Economic Cooperation and Development, Paris


October



ANNEX A: EPIDERMTM SKIN IRRITATION TEST: FLOWCHART

Transfer tissues from agarose to assay medium

Incubate (37°C, 5% CO2) for 1 hr

Transfer tissues to fresh assay medium

Dose 3 tissues each with 25 µL / 25 mg TS, or PC, or NC

Expose at room temperature tissues

Stop exposure after 15 minutes by rinsing with PBS

Transfer tissues to fresh assay medium

Incubate (37°C, 5% CO2) for 42 hours

Collect medium for IL-1 analysis

Blot tissues and transfer them into MTT medium

Incubate (37°C, 5% CO2) for 3 hours

Aspirate MTT medium, rinse tissues with PBS, blot

Immerse the inserts in extractant solution (isopropanol)

Extract formazan 2 hours (shaking, room temperature)

Equilibrate extracts from beneath and atop tissue

Read OD in a plate spectrophotometer at 570 nm


October



ANNEX B: METHODS DOCUMENTATION SHEET (MDS)

Assay No:...............................................

Date:.......................................................

Corresponding XLS data file name:........................................................................

Performed by:...........................................................................................................

TIME PROTOCOL

Receipt of EpiDerm tissues (date, hour): ....................................................................................

ID/ date:

Experimental schedule procedure preincubation

(1 hour)

exposure & washing

(15 min + 15min)

incubation

(42h)

MTT assay

(3h)

formazan extraction

(2h or overnight)

start stop start stop start stop start stop start stop

ID/ date:

Measurement (date, hour): ...............................................................................

ID/ date:


October



DEVICES VERIFICATION

Incubator verification

Incubator #

CO2 < 5% ± 0,5 % >

Temperature < 37°C ± 1°C >

Check water in reservoir ( )

ID/ Date:

Refrigerator verification Water bath verification

Refrigerator # Temperature

< 5°C ± 2°C >

Water bath # Temperature

< 37°C ± 1°C >

ID/ Date: ID/ Date:

In case that your devices are controlled by central computer, fill in the following table instead

of fields above:

Name of the device device # reference

ID/ Date:

Pipette verification (triplicate weightings)

Pipette 3 x H2O into a small baker on a laboratory scale and record readings in g. Perform pipette veri-fication only once per week and refer to it in all assays of this week. If adjustable pipettes are used, check adjustment daily.

0.9 mL 2 mL 300 µL 200 µL 25 µL positive displacement pipette 25 µL

..........................................................H2O weight in g......................................................

1.

2.

3.

Mean

SD

ID/ Date:


October



KIT RECEIPT

EpiDerm kit received (day/date/hour):

Day used:

EpiDerm (EPI-200) Lot no.: No. of inserts:

Production date:

Assay medium ( EPI-100-ASY) Lot no.:

Expiration date:

MTT concentrate (MTT-100-CON); 2 ml Lot no.:

Expiration date:

MTT diluent (MTT-100-DIL); 8 ml Lot no.:

Production date:

MTT extractant (MTT-100-EXT), 60 ml Lot no.:

Expiration date:

PBS (TC-PBS); 125 ml Lot no:

Expiration date:

No. of 6 well plates:

No. of 24 well plates

MTT protocol + packaging diagram

Position of Ice-packs: (direct contact of the ice-packs with the skin must be avoided)

Other remarks

ID/ Date:


October



QUALITY CONTROL OF THE SKIN Use scores: 1- very good, 2-good, 3- acceptable, 4- not acceptable

APPERANCE KIT 1 KIT 2 KIT 3 KIT 4

MACRO.

MICRO

OTHER as. -edge defects -air bubbles -wet tissues --

If you have any special observations (listed in OTHER), make reference to related tissue(s)

Kit No.1:......................

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24

Kit No.2:......................

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24

Kit No.3:......................

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24

Kit No.4:......................

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24

ID/ Date:


October



SOLUTIONS

POSITIVE CONTROL SDS 5% solution in distlilled stérile water (w/v) :

- SDS reference, batch N°:……………………………………………………….

- Weight: ………………………………………………….….................................

- Distilled water, volume added:………………………………………………….

- Preparation date: ……………...………………………………………………..

- Expiration date : ………………..……………………………………………….

- Stocking place : Refrigerator N°..…………………………………………........

Note: In case that if you are preparing your own MTT stock solution and/or PBS fill in the folowing forms

MTT stock solution preparation: 5 mg/ml :

- MTT batch N° : …………………………………………………………….…..

- Weight : ………………………………………………………………………..

- PBS batch N°: …………………………………………………………………

- PBS Volume added: …………………………………………………………….

- Preparation date: ………………………………………………………………

- Expiration date : ……………………………………………………………….

- Stocking place : Refrigerator N°…………………………………………........

PBS solution preparation:


- pH adjustment………………………………………………………………......

- Type of sterilisation...........................................................................................


- Expiration date : ……………………………………………………………….


October



DOSING PROCEDURE

Tick ( ) in the columns the type of application (pipette, patch, spoon...)

Application (25 mg / 25 µl) Code Liquid/ Solid

(L/S) Pipette

( )

Mesh

( )

Spoon

( )

Pin

( )

Wetting

( )

Remarks

(P:pasty; V: viscous; W:waxy; ST:sticky; C:creamy) ID/ Date:


October



REMARKS TO SINGLE TISSUES

If you will during the assay observe any abnormality, fill in following table. Record the tissue number, substance code and your observation or remark.

Tissue No.

Substance code

Remark

ID/ Date: SPECTROPHOTOMETRICAL MEASUREMENT


October



Note: switch on the reader 15 min before reading

Check plate photometer filter Tick correct ( ) filter setting reading filter: 570 nm no reference filter

ID/ Date:

PLATE CONFIGURATION FOR READING (for transfer to I-Spread) :

Record the positions of substances on 96-well plate

PLATE 1 PLATE 2 PLATE 3

1 NC 1 1

2 PC 2 2

3 3 3

4 4 4

5 5 5

6 6 6

7 7 7

8 8 8

9 9 9

10 10 10

11 11 11

12 12 12

ID/ Date:

ARCHIVATION

Raw data saved in/as:

Spreadsheet saved in/as:

MDS saved in/as:


October



ANNEX C: CHARACTERISATION OF TEST SUBSTANCES

Remarks Code Physical state (S/L)

Colour

MTT

redu

c-tio

n (

+ / -

)

Rea

ctio

n w

ith m

esh

(+

/ - )

known R-phrases

known S-phrases

special storage requirements

other


October



SOLID SUBSTANCES ONLY

Wight of substance (when applied by spoon) in mg

Code

1. 2. 3.

Grinding

(yes / no)

Remarks

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