© ECVAM DB-ALM: Protocol

DB-ALM Protocol n° 131 : EpiSkin™ Skin Irritation Test15min - 42 hours
Skin Irritation and Corrosivity

The irritation potential of a chemical is predicted through its cytotoxic effect on the Reconstructed
Human Epidermis (RHE) model EpiSkin T M , which is measured by the MTT cell viability assay. The test iscompliant with the OECD Test Guideline No. 439 - In Vitro Skin Irritation: Reconstructed Human
Epidermis Test Method.
Objective & Application
TYPE OF TESTINGLEVEL OF ASSESSMENTPURPOSE OF TESTING:::

Replacement, screening
Toxic potential, hazard identification
Classification and labellingContext of Use:
The test method is based on the Reconstructed Human Epidermis (RHE) model, which was granted regulatory
approval as a full replacement for the rabbit in vivo skin irritation test under the EC Test Method Regulation(Method B.46, EU, 2009) and under conditions laid down in OECD Test Guideline 439 (OECD, 2010).The test
method can be used for the hazard identification of irritant chemical substances and mixtures in accordancewith the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS,
UN, 2009) and European Commission Regulation on the classification, labelling and packaging of substancesand mixtures (CLP, EC No.1272/2008, EU, 2008). Positive results obtained with the test method enable theclassification of test substances as "Category 2: Irritant". If the regulatory authorities do not require the optional
UN GHS classification of "Category 3: Mild irritant", remaining test substances are labelled as “No Category”(Method B.46, EU 2009; OECD TG 439, 2010).The test method can be used within the sequential dermal
irritation/corrosion testing strategy for the full characterization of skin irritation potential (OECD TG 404, 2002;
Method B.4, EC No. 440/2008, EU 2008).The EpiSkin T M SKIN IRRITATION TEST 15min - 42 hours was the first
protocol based on the Reconstructed Human Epidermis (RHE) to be validated and is therefore designated as
Validated Reference Method (VRM) in OECD TG439.
Applicability Domain:
Solids (soluble or insoluble in water)
Liquids (aqueous or non-aqueous)
Semi-solids
Waxes
The method is not designed for testing of gases and aerosols or highly volatile test compounds.Highly coloured
chemicals and/or MTT reducers may interfere with cell viability measurements and therefore adapted controls
for corrections need to be used.The Validated Reference Method EpiSkin T M SKIN IRRITATION TEST 15min - 42hours represents a performance standard for the predictive capacity of other RHE-based methods. The
performance of any novel method must be comparable or better than that of VRM (EpiSkinT M ) against a definedtest set of 20 reference test chemicals (OECD TG 439, 2010).

Résumé
Skin irritation refers to the reversible damage to the skin following the application of a chemical for up to
4 hours, as defined by the United Nations Globally Harmonized System of Classification and Labeling ofChemicals (UN GHS, UN, 2009). The potential to induce skin irritation is an important consideration forprocedures for the safe handling, packing and transports of chemicals.
Skin irritation in vivo is determined by a modification of the original Draize rabbit skin irritation test
(Draize et al., 1944) as described in the OECD TG 404 (OECD, 2002).
In 1998 an ECVAM workshop made an inventory of the various methods for in vitro assessment of theirritant potential of chemicals (van de Sandt et al., 1999) and a scientific validation of related methods
was undertaken (Botham et al. 1998). During the pre-validation steps, several methods and subsequent
refinements were proposed (Zuang et al., 2002; Portes et al., 2002; Cotovio et al., 2005) and finally threemethods were accepted for further validation process. To facilitate future regulatory acceptance, acommon Prediction Model and protocol, differing only by model-specific details, were agreed to be usedwith both EpiSkin T M (EPISKIN SNC) and EpiDerm T M (MatTeK Corp.).
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The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis(RHE) model, followed by a cell viability test. Cell viability is measured by the mitochondrial
dehydrogenase conversion of MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into
a blue formazan salt that is quantitatively measured after extraction from tissues (Mossman, 1983). The
reduction of the viability of tissues exposed to chemicals, in comparison to negative controls (treatedwith Dulbecco's D-PBS), is used to predict the skin irritation potential.
Experimental Description
Endpoint and Endpoint Measurement:
TISSUE VIABILITY: determined by a reduction in mitochondrial dehydrogenase activity, measured byformazan salt production from MTT. It is expressed as % of the negative control
Endpoint Value:≤ 50% VIABILITY: ≤ 50% reduction in cell viability is a cut-off value for the presence of a significant
biological effectExperimental System(s):RHE EpiSkin T M : Small Model (EpiSkinT M -SM, EPISKIN SNC Lyon, France) is a three-dimensional
human epidermis model. Human-derived epidermal keratinocytes are seeded on a dermal substituteconsisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified
epidermis model is obtained after 13-day culture period comprising the main basal, supra basal,
spinous and granular layers and a functional stratum corneum (Tinois et al., 1994).
Basic Procedure
Test chemicals are applied topically to the epidermal model for 15 minutes at room temperature. Threeepidermis units are used per each test chemical, positive and negative controls. Exposure to the test
chemical is terminated by rinsing with phosphate buffered saline (PBS). Epidermis is then incubated at
37°C for 42 additional hours. For each test chemical, three independent tests with three different batchesof EpiSkinT M are normally used. Aliquots of culture media may be collected and kept frozen (-20°C) for
cytokine (IL-1alpha) measurements.
The cell viability is assessed by a subsequent incubation of the tissues for 3 hours with MTT solution in a
12 well plate. The precipitated formazan is then extracted using acidified isopropanol and quantified
spectrophotometrically at 570 nm using 96 well plates.
Optional step: the protein levels of IL-1alpha in the supernatant may be determined and reported in
pg/ml, as an additional information to the TG439's viability endpoint.
SDS 5% and PBS-treated epidermis are used as positive and negative controls respectively. For eachtreated tissue the viability is expressed as a % relative to negative control tissues (mean).
Data Analysis/Prediction Model
Irritation potential of a test chemical is determined according to the UN GHS and EU CLP as "Category 2:
Irritant or “No Category” (UN, 2009; No. 1272/2008, EU, 2008). Category 2: Irritant is predicted if themean relative tissue viability of three individual tissues exposed to the test chemical is reduced below
50% of the mean viability of the negative controls. The prediction model is defined as described below:
In vitro results

In vivo classification

Mean tissue cell viability ≤ 50%

Category 2:Irritant

Mean tissue cell viability > 50%

No Category

Test Compounds and Results Summary
A total of 58 test compounds, consisting of 25 in vivo irritants and 33 in vivo non irritants, according to
the European classification system based on the Dangerous Substance Directive (EU DSD, EU, 2001),
have been tested in the ECVAM Skin Irritation Validation study (SIVS, 2003 - 2007). The results wereinterpreted on the basis of a fixed 15 min exposure-time and 42 hours post-exposure incubation timehttps://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocols

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© ECVAM DB-ALM: Protocol(ESAC, Meeting 30, 2009), according to the EU DSD classification, and the UN GHS (UN, 2009) and its EU
implementation (CLP, No. 1272/2008 , EU, 2008).
EU DSD

UN GHS / EU CLP

EpiSkin (MTT)*

EpiSkin (MTT)*

Sensitivity

72.0%

84.6%

Specificity

81.8%

71.1%

Accuracy

77.6%

74.1%

* Data from the "Explanatory Backgound Document to the OECD Draft Test Guideline on in vitro Skin
Irritation Testing", 2009, available at:/cm_esac/ESAC31_skin-irritation-statement_20090922.pdf
Acceptance Criteria and Proficiency Testing
A generic description of general and functional conditions that human skin models need to comply with,can be found in the OECD Test Guideline 431 In vitro Skin Corrosion: Human Skin Model test (OECD,
2004).
In OECD TG 439 (OECD, 2010), it is recommended, prior to a routine use of the test, to check thetechnical proficiency of the laboratory using ten Proficiency Chemicals listed in the Table below:
Chemical

CAS NR

In vivo score
OECD TG 404

Physicalstate

UN GHS Category

naphthalene acetic acid

86-87-30

Solid

No Cat.

isopropanol

67-63-00.3

Liquidmethyl stearate112-61-8

1Solid

heptyl butyrate

5870-93-9

1.7

Liquidhexyl salicylate6259-76-3

2cyclamen aldehyde

103-95-7

2.3

1-bromohexane

111-25-1

2.7

potassium hydroxide (5% aq.)

1310-58-3

3

Cat. 2

1-methyl-3-phenyl-1-piperazine 5271-27-2

3.3

Solid

heptanal

3.4

Liquid111-71-7

No Cat.
(Optional Cat. 3*)* Classification of a test chemical under the optional UN GHS Category 3: "Mild Irritant" is currently notpossible with the EpiSkin TM test and not required under EU CLP regulation (No. 1272/2008 EU, 2008).
Discussion
The EpiSkin T M test relies on the MTT reduction as a quantitative indicator of cell viability. The protocol
includes several check methods developed to detect and compensate for the properties of a test
chemical, which may interfere with the accuracy of the test. Some chemicals can directly reduce the MTT
reagent, other chemicals can directly color the tissue or the cells. These properties of test chemicals caninterfere only if sufficient amounts of the chemical are still present in (or onto) the tissue at the end of
the 42 hours post-treatment incubation period. In these cases, a special procedure for the quantificationof the genuine MTT mitochondrial reduction should be applied. The use of specific controls enables thequantification of the bona fide tissue viability. After subtracting the unspecific Optical Densitiy due to
direct chemical MTT reduction and/or to chemical residual color extracted from the tissues, theremaining OD570nm is used to calculate the cell viability %. Conditions for performing specific control
steps are described in the Procedure Details section of this protocol.
StatusParticipation in Validation Studies:
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Upon review of existing information by the ECVAM Skin Irritation Task Force and an ECVAM Workshop
both EpiSkin T M and EpiDermT M skin irritation (SIT) tests were regarded as sufficiently promisingpredictors for skin irritancy potential and ready to enter the formal validation study (Portes et al., 2002
and Cotovio et al., 2005).
Following the successful conclusion of the ECVAM Skin Irritation Validation Study (SIVS, 2003 – 2007),the ECVAM Scientific Advisory Committee (ESAC) endorsed the scientific validity of EpiSkin T M method
as a stand-alone replacement for the Draize Skin Irritation test for the purpose of distinguishing between
skin irritating and non-skin irritating substances (ESAC, 2007 and ESAC, Meeting 30, 2009).
Following the 26 t h meeting of the non-Commission members of the ECVAM Scientific Advisory
Committee, performance standards for applying human skin models to in vitro skin irritation testing
were defined based on the validated test EpiSkinT M method (ESAC, 2007). These performance standards
were revised after implementation of UN GHS (ESAC, Meeting 31, 2009) and can be used to evaluate theaccuracy and reliability of other analogous test methods (also referred to as "me-too" tests) that are
based on similar scientific principles and measure or predict the same biological or toxic effect.
Regulatory Acceptance:
In July 2009, the reconstructed human epidermis test methods for skin irritation testing which meetcertain criteria (such as EpiSkinTM , EpiDermTM SIT and SkinEthicTM RHE) have been included as
Method B.46 of the Annex to 440/2008/EC (EU Test Methods Regulation; EU, 2008) during its 1st
adaptation to technical progress (Commission Regulation No 761/2009/EC; EU, 2009).
In 2010, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method was adopted as theOECD Test Guideline No 439 which is applicable to the assays for skin irritation employing
reconstructed human epidermis models (EpiSkin TM , EpiDermTM SIT and SkinEthicTM RHE) (OECD,2010). OECD TG 439 defines functional and performance criteria for other (or new) skin or epidermis
models used in the context of this guideline (OECD, 2010).
Health and Safety Issues
General Precautions
The epidermal cells are taken from healthy volunteer donors negative to anti-HIV 1 and 2, and to
hepatitis C antibodies, and to hepatitis B antigens. Nevertheless, normal handling procedures forbiological chemicals should be followed:(a) It is recommended to wear gloves and glasses during handling;(b) After use, the epidermis, the chemical and all media in contact with it, should be decontaminated (forexample, by using a 10% solution of bleach or appropriate containers), prior to disposal.
Examine all kit components for integrity. If there is a question, a concern or something unusual call:
EPISKIN SNC at: +33 (0)4 37 28 72 00 for technical support, and for future users: www.skinethic.com.
Safety instructions for working with chemicals:
Store chemicals in ventilated safety cup boards. Respect special store conditions if necessary (special
temperature, protected from light etc.)
Non-coded chemicals should be handled following chemical safety datasheet.
Unknown and coded chemicals with none or incomplete safety handling information should beconsidered as irritating and toxic and must be handled with maximum care. In accordance withchemical safety guidelines: use safety ventilated cabinet, wear gloves, eye and face protections.
MTT( R26 R68 R22 R36 R37 R38), isopropanol (R11 R36 R67), and HCl (R34 R37) are dangerous. Workin ventilated cabinets, to prevent accidental contact wear protective gloves, and if necessary a mask
and/or safety glasses.
Abbreviations and Definitions
CLP: Classification, Labelling and Packaging
DSD: Dangerous Substance Directive
D-PBS: Dulbecco's Phosphate-Buffered Saline
ECVAM: European Centre for the Validation of Alternative Methods. As from 2011, ECVAM has been
established as the European Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM),
hosted by the Joint Research Centre, Institute for Health and Consumer Protection (IHCP).
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ESAC: ECVAM Scientific Advisory Committee
EU: European Union
EC: European Comission
IC50: Concentration that induce 50% decrease in cell viability
I: Irritant
IL-1alpha: Interleukin-1alpha
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NC: Negative Control
NI: Non Irritant
OD: Optical Density
OECD: Organization for Economic Co-operation and Development
PBS: Phosphate Buffered Saline
PC: Positive Control
RHE: Reconstructed Human Epidermis
SDS: Sodium Dodecyl Sulphate
SIVS: Skin Irritation Validation Study
SIT: Skin Irritation Test
TG: Test Guideline
UN GHS: United Nations Globally Harmonized System of Classification and Labelling of Chemicals
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PROCEDURE DETAILS, 20 June 2011
EpiSkin™ Skin Irritation Test 15min - 42 hours
DB-ALM Protocol n° 131

The protocol is compliant with OECD TG 439 In Vitro Skin Irritation: Reconstructed Human Epidermis
Test Method.
Some figures in this protocol are snapshots of the actual calculation sheets used for data analysis and areprovided here for illustration only. Online version of the protocol includes editable version of MDS
sheets, to be filled in when compiling the experiment records. Go to DB-ALM webite(http://ecvam-dbalm.jrc.ec.europa.eu/), section related to protocol No. 131 and select Related
information: Downloads.
Contact Details
Dr. Nathalie Alépée
Research & Innovation
L'ORÉAL
1 Avenue Eugène Schueller
Aulnay sous Bois 93600
Franceemail: nalepee@rd.loreal.com
telephone: +33148689102
Dr. José Cotovio
Director
Research & Innovation
L'ORÉAL
1 Avenue Eugène Schueller
Aulnay sous Bois 93600
Franceemail: jcotovio@rd.loreal.com
telephone: +33148689102
Materials and Preparations
Cell or Test System
The EpiSkin T M -SM kit and biopsy punch can be obtained from EPISKIN SNC, 4 Rue Alexander Fleming– 69366 Lyon cedex 07 France.
Phone: +33 4 37 28 72 00
Fax: +33 4 37 28 72 28
http://www.skinethic.com/order.asp
Equipment
Fixed Equipment
Item

Use

Microbiological safety cabinet (laminar flowhood)

For safe work under sterile conditions

Non-sterile ventilated cabinet

For safe work with chemicals, applications, washes

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

For incubating tissues

Plate reader (96 well) with a 570 nm filter

For optical density readings (MTT)

Water bath at 37°C

For warming media

Vacuum source/trap and Pasteur pipettes

For aspirating media and solutions

Laboratory balance (accuracy 0.1 mg)

For checking chemicals weight and spatula weight

Shaker for 12 well plates

For shaking tissues before media sampling

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1 glass funnel

For dropping wash fluids in the bottle

Wash bottle (500 ml )For collecting wash fluids

1 gauged flask

For SDS 5% solution preparation

200 ml beakers

For PBS (washes)

Adjustable micro-pipette – 0 to 200 µl

For pipetting 200 µl formazan extracts (96 well plate)

Adjustable multi-step pipette, 25 ml combitips

For assay and maintenance media (2 ml), washing

Adjustable positive displacement micro-pipette 0 to 25 µl

For application of liquids and semi-solids 10 µl

Mortar and Pestle

For grinding granulars

Stop-watches/Timers

For controlling contact and step times

Small sterile blunt-edged forceps

For handling tissue inserts

Small sterile flat-ended forceps

For separating the epidermis from the collagen

Vortex mixer

For shaking MTT solution and SDS 5% solution

Small curved flats spatula

For weighing and spreading solids and/or sticky
chemicals

1x EpiSkinT M -SM biopsy punch (EPISKIN SNC)

For separating the tissue from the insert, prior toisopropanol extraction

Consumables
Item

Use

Sterile bottle (eg. 250 ml)

For diluting MTT in the assay medium

Conical opaque centrifuge tube - 50 ml

For MTT stock solution preparation

Small glass weight boats

For weighing powders

Conical “safelock” micro-tubes (1.5 ml) or
equivalent

For formazan extraction

96-well flat-botom plates

For reading Optical Density

12-well sterile plates

For tissues incubation and application steps (1 plate
per test-chemical)

Nylon mesh (e.g., Sefar Nitex 6 mm, cat.No.
03-150/38)

For weighing and spreading sticky chemical

Cryovials- polypropylene

For collecting and freezing media samples for eachtissueCotton-tipped swabs

For drying the tissue surface

Media, Reagents, Sera, others
The EpiSkinT M -SM kit components:
Description

Use/Comment

1 x EpiSkinT M -SM plate containing 12
reconstructed epidermis units (area: 0.38 cm2
each)

Each reconstructed epidermis is attached to the baseof a tissue culture insert with an O-ring set andmaintained on nutritive agar for transport

1 x 12-well assay plate

For assays

1 x flask of sterile maintenance medium

Basic medium for incubations

1 x flask of sterile assay medium

Basic medium for use in MTT assays

Materials not provided with the EpiSkinT M -SM kit:
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Item

Use

12N HClFor pH adjustment of isopropanol

Isopropanol

For formazan extraction

MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide](e.g., Sigma cat.No. M2128)

For viability measurements

5 % SDS [151-21-3] (w/v; aq; e.g., Sigma, cat.No.
L4509, purity 99%) in sterile distilled water

To be used as positive control in each assay

Dulbecco’s D-PBS, GIBCO 14040-091 orequivalent

For rinsing tissues and negative control

Sterile distilled H2O

For powder applications

Preparations
Media and Endpoint Assay Solutions
MTT solution preparation:
Note: MTT solution is light sensitive. Protect it from light using silver paper.
(a) MTT stock solution preparation
Prepare a 3 mg/ml solution in saline buffer (PBS)
Vortex 15 minutes
Keep at 4°C protected from light for up to 15 days in the refrigerator.
(b) MTT ready to use solution preparation
Pre-warm assay medium to 37°C
Dilute stock solution 1v+9v, with assay medium (final concentration 0.3 mg/ml)
Protect from light until use (do not exceed 3 hours storage before use)
Fill MDS Annex 8
5% (W/V) SDS (aq) preparation
Weigh SDS and add distilled water in order to reach the final concentration of 5% (w/v), i.e., 1 g ofpure SDS qsp 20 ml water using a gauged flask.
Preparation can be stored at 4°C for one month.
Fill MDS Annex 8
Acidic isopropanol
Prepare acidic isopropanol (final concentration: 0.04 N HCl )Dilute 1.8 ml of 12 N HCL in 500 ml of isopropanol.
Preparation can be stored at 4°C protected from light (silver paper) for one month.
Fill MDS Annex 8
Water- killed epidermis preparation (for MTT-interacting substances)
Place the living epidermis in a 12 well plate with 2 ml of distilled water (replacing the culture
medium).
Incubate at 37°C, 5% CO2, in a >95% humidified atmosphere for 48 +/- 1 hours.
At the end of the incubation, discard the water.
Keep dead epidermis frozen (dry) in freezer at -18°C to -20°C (dead epidermis can be stored and used
up to 6 months).
Tissues should be de-frozen before use at room temperature (1 hour in 2 ml of “maintenance”
medium).
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Further use of thawed tissues is similar to living tissues.
Test Compounds
Main information concerning the chemicals (codes or numbers, physical consistence, volumes orweight, expiration date, storage conditions, etc.) should be registered in the MDS Annex 3.
Application of Test Chemicals
This is a detailed description of how to apply test chemicals onto the test system. For complete experimental
setup see the Test Material Exposure Procedures section (p.12).
Liquids
Apply 1 0 µ (26.3lµl/cm2) on the top of each epidermis, using a positive displacement
pipette.
Gently spread it with the pipette onto the epidermis surface. Ensure to cover all thesurface. Record in the MDS.
Solids
The chemical should be crushed to a fine powder, if necessary, using a mortar and apestle.
Apply first 5 µl of distilled water using a positive displacement pipette to the epidermal
surface in order to improve further contact between the powder and the epidermis.
Gently spread with the pipette.
Apply 10 mg +/- 2 mg (26.3 mg/cm2) of the powder to the epidermis surface. (usespecial glass weigh boats or similar tools to avoid electrostatic electricity).
Gently spread on the epidermal surface with a curved flat spatula.
Record details in MDS Annexes 5, 6, 7 if necessary (grinding, spreading and volume of
PBS used)
Viscous and sticky chemicals
Use a curved flat spatula and weigh directly the chemicals sticked on the curved edge ofspatula. In order to spread 10 mg +/- 2 mg (26.3 mg/cm2) onto the epidermis surface,weigh a higher amount (12 + 2 mg) to compensate for the product remained on the
spatula. Gently spread onto the surface by repeated circular movements in order tocover the all surface. A nylon mesh can be used for spreading sticky chemical.
Record details: note spatula weight before application and after spreading in the MDS 5
and 6.

Curved flat micro spatula

Normal supplied stainless steel spatulas can be“home adapted” by twisting the end of the roundedpart of the spatula.
Maximum and minimum size of the flat (see below)
should be respected. Edges should be blunt in order toavoid tissue damage during spreading.

Check-method for possible direct MTT reduction with test substancesThis verification should be done before starting the experiment.
Relative conversion of MTT by the tissue is the key
parameter evaluated in this assay. Therefore, it isnecessary to assess the non-specific reduction of MTT
by the test substance used.
Prior to experiments all test substances should be putin contact with the MTT solution as described below.
Fill each well of a 12 well plate with 2 mL of
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Fill each well of a 12 well plate with 2 mL of
MTT solution (0.3 mg/mL).
Add 10 µl or 10 mg of the substance to beevaluated or water for control and mix.
Incubate the mixture for 3 hours at 37° C
protected from light (test conditions).
If the MTT solution color becomes blue or purple, thesubstance interacts with the MTT. It is then necessary
to evaluate during the future studies the part of OD
due to the non specific reduction of the MTT (i.e. byusing killed epidermis or water killed epidermis).

A: control
B: chemical 1 - no interaction
C: chemical 2 - slight interaction
D: chemical 3 - strong interaction
Fill the MDS annex 4
Check-method to detect the coloring potential of test-substancesThis verification should be done before starting the experiments
Prior to treatment, chemicals should be evaluated for their intrinsic color or ability to become colored incontact with water, simulating a humid environment of the epidermal tissue. Add 10 mg (solids) or 10 µl(liquids) of the test-chemical to 90 µl of water in a transparent recipient (micro-tube). Mix for 15 minutes.
At the end of the shaking period color check is performed by the operator (unaided eye assessment). If acolored solution is detected, the tissue staining ability of the test-chemical needs further check (go to thenext step: Check-method to detect coloring test-substances ability to stain tissues). Otherwise, thetest-chemical is evaluated as a common chemical. See Test Material Exposure Procedures section forfurther instructions.
Check-method to detect coloring test-substances ability to stain tissues
This verification should be done before starting the experiments
Prior to treatment, colored chemicals should be evaluated for their ability to color and stain the tissues inthe test conditions. The non specific color (NSC) is first quantified by using one living tissue unit per
chemical. Apply 10 mg (solids) or 10 µl (liquids) of the test-chemical onto the tissue and run a PBS
control in parallel. Follow all steps described in chapters Preparation and pre-incubation (Day 0) to
Absorbance / Optical Density measurements (Day 3 or 6), but with only one live tissue unit for eachtest-chemical. Replace the MTT incubation period by incubation with the assay medium without MTT.
If the test chemical-linked optical density (unspecific OD570) is >5% and ≤ 30% relative to the
PBS-negative control OD570, the chemical can be evaluated in further trials by using additional controls
in all runs in order to correct final values (subtract viability unrelated OD).
Test-chemicals with NSC ≤ 5% relative to the negative control should be evaluated by using the common
procedure. If non specific color NSC is > 30% relative to the negative control, additional steps must be
undertaken or the chemical must be considered as incompatible with the test.
Positive Control(s)5% SDS (w/v; aq) is used as positive control in each assay.
Negative Control(s)Dulbecco’s D-PBS is used as negative control.
MethodTEST METHOD QUICK WORK FLOWCHART
1. Check test-chemicals for their potential direct MTT reducing activity
2. Check the presence of dyes and chemicals able to color the epidermis
3. Receipt: Transfer epidermis from transport medium to maintenance medium
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4. Incubate for 24 hours (37°C, 5% CO2, humidified atmosphere)
5. Apply topically 10 µl or 10 mg of Test Chemical or PC or NC
6. Keep at room temperature for 15 minutes
7. Stop treatment by rinsing with PBS
8. Transfer tissues to fresh maintenance medium
9. Incubate 42 hours (37°C, 5% CO2, humidified atmosphere)
10. Sample medium for mediators release (optional)
11. Transfer tissues to MTT solution
12. Incubate tissues for 3 hours (37°C, 5% CO2, humidified atmosphere)
13. Make biopsy punch of the tissues
14. Transfer and immerse in acidified isopropanol
15. Formazan extraction: 4 hours (room temperature) or 48-70 hours (at 4°C) one weekend
16. Shake and homogenise
17. Transfer extracted solution in 96 well plate
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Test System Procurement
For contact details on purchasing the kit -see Materials and Preparations section.
The EpiSkinT M -SM kit contains 12 reconstructed epidermis units and the necessary culture media(maintenance and assay media) and additional 12 well plates. Each EpiSkinT M -SM batch iscontrolled by the manufacturer. Results of quality controls are supplied with the kits.
Routine Culture Procedure
Receipt of the EpiSkinT M -SM kits (Day 0)
Upon arrival, register kit details (reception etc.) and assay procedures on the MDS form. (in MDS Annex
2)
Check the date of sending written on the package as well as some critical points before opening the
EpiSkinT M -SM kit:inspect the color of the agar medium used for transport and check that its pH is acceptable:orange color - acceptable; yellow or violet color - not acceptable;
inspect the color of the temperature indicator to verify that the kit has not been exposed to atemperature above 40°C:
pale grey - acceptable; dark grey - not acceptable;
Place the assay medium supplied with the kits at 2-8°C (refrigerator).
Pre-warm the maintenance medium supplied to 37°C (water bath).
Leave the EpiSkinT M -SM kits in their packaging at room temperature in the microbiological safetycabinet until the next step.
Preparation and pre-incubation (Day 0)This step should be conducted in sterile conditions (Microbiological safety cabinet)
Note: One sterile 12 wells plate is used for one test-chemical. For a given chemical, the same plate will be
used for all steps of the protocol. Prior to epidermis transfer from their transport packaging, prepare
plates labeling. Mark all plate lids with the code number of the test-chemical (3 wells per chemical), or
negative control (3 wells) or positive control (3 wells).
(a) Fill 3 wells of the first column of a 12
well assay plate with pre-warmed
maintenance medium ( 2 ml perwell, adjustable multi-step pipette).

(b) Open the EPISKINT M -SM kits andtransfer the epidermis units into
maintenance medium filled wells,
using sterile forceps. Use 3 units perplate.
Avoid air bubbles formation under thetissue.
Label inserts by replicate treatment
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Ex: Rep 1; rep 2; rep 3(c) Incubate at 37°C, 5% CO2, in a >95% humidified atmosphere for at least 24 hours.
Test Material Exposure Procedures
Important: Some highly reactive chemicals can produce fumes which may affect adjacent units in thesame plate. It is strongly recommended to test all unknown chemicals alone in separate plates. It isalso recommended to routinely incubate positive and negative controls in separate plates. Never use
cotton buds for spreading the substance onto tissues.
Application of the test substance and rinsing (Day 1)
Controls for MTT direct-interacting chemicals
For each MTT-interacting substance previously detected, and in addition to the normal procedure, 3
killed treated tissues and 3 killed negative control tissues should be used for the MTT evaluation in
one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associatedactivity).
All killed tissues must be both from the same batch. The batch of killed tissues can be different thanthe batches of the living tissues. These tissues should follow the same treatment steps than the living
tissues.
Controls for dyes and chemicals able to color the tissue
For each substance previously detected as being able to color the tissue, and in addition to thenormal procedure, at least 1 additional chemical-treated tissue and at least 1 additional negative
control per run should be used for the non specific OD evaluation.
These tissues should follow the same treatment steps than other tissues except for the MTT step:
MTT incubation is replaced by incubation with fresh assay medium. OD readings are made following
the same conditions then other tissues.
Fill MDS Annex 6

Specific procedure for the positive control (5%SDS)
Apply 10 µl with the positive displacement pipette. To ensure a correct contact with the entire
epidermis (particularly the center), spread the solution directly with the pipette tip.
Continue spreading for approximately 30 seconds, to ensure that the SDS covers all the epidermis, i nparticular the center.
Preparation(a) Pre-warm the maintenance medium to 37°C (water bath).
(b) Fill the second column (3 wells) of a 12
well assay plate with pre-warmed
maintenance medium ( 2 ml per well,
adjustable multi-step pipette). Verify theplate lids labeling (code number of thetest-chemical (3 wells per chemical), or
negative control (3 wells) or positive control(3 wells).

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Topical applications: 15 minutes treatment
Note: chemicals are applied undiluted (neat)
Apply 10 µl or 10 mg of the undiluted chemical (or negative control or positive control) on the top of
the epidermis. 3 tissues per chemical should be used and follow the defined application order(replicate1, replicate 2, replicate 3).
Keep 1 minute (example) interval between each tissue application, and do not test more than 4chemicals (=12 tissues) in a block in order to be ready to wash off the test chemical in time (15minutes contact).
Record on the plate lid the exact timings during the experiment and fill the corresponding MDS
Annex 7 at the end of each run.

Positive displacement pipette deposit.

Spatula spreading (viscous, waxy … )Keep the plate (lids on) containing the treated epidermis for 15 minutes ( ± 0.5 minute) in theventilated cabinet at room temperature (comprised between 19°C to 23°C).
End of the treatment and removal of the test chemical
After 15 minutes exposure (+/- 0.5 min):(a) Remove the treated units using forceps, and rinse thoroughly with 25 ml sterile PBS (ready to use)filling and emptying the tissue inserts, to remove all residual test chemical from the epidermal surface.

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© ECVAM DB-ALM: Protocol(b) Place the units on an absorbent paper, remove the remaining PBS from the epidermal surface by
gently taping, and sweep the surface with a cotton-bud if necessary without damaging the epidermis.

(c) Transfer the blotted tissue units in the
new maintenance medium pre-filled wells(2n d column).

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Post treatment Incubation: 42 hours
Incubate the treated and rinsed epidermis at 37°C, 5% CO2, 95% humidified atmosphere for 42 hours± 1 hour. Record incubation start time in the MDS Annex 9.
Recommended media sampling for mediators and enzyme release measurement (optional)
At the end of the 42 +/- 1 hour incubation period (Day 3):
Before starting, label an appropriate numbers of polypropylene tubes with caps (one per tissue unit):
batches number, chemicals code number, replicate number and date.
Out of the incubator, shake the plates containing the treated epidermis (lids on) on a plate shaker for
15 minutes +/- 2 min, medium speed (300 rpm/min). This step helps to homogenise the released
mediators in the medium before sampling.
Transfer 1.6 ml of incubation medium from each tissue to the pre-labeled tubes. Stock Frozen at- 20°C until analyses (for at least one year).
Note: This optional step is not included in TG439. The procedure allows for the collection of additional
information on the response of the test system to the chemical tested. Please refer to the contact personsof this protocol for more details of the relevant auxiliary measurements.
Endpoint Measurement
MTT test after the 42 hours incubation period (Day 3)(a) Fill the appropriate number of wells of
the assay plate (3 rd column) with 2 ml per
well of 0.3 mg/ml MTT in assay medium.

(b) Transfer the EpiSkinT M units to the MTT
filled wells:
Sweep excess medium on the unit
bottom with absorbent paper
before setting down on the well.
Replace the lid on the plate.

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© ECVAM DB-ALM: Protocol(c)Incubate for 3 hours ( ± 5 minutes) at 37°C,
5% CO 2, 95% humidified atmosphere.
Record start time in the MDS Annex 9.
Note: Viable cells metabolize MTT. The blue
color of the epidermis is due to theintracellular formation of formazan crystals.

(d) At the end of the incubation, record observations and comments in MDS Annex 10.
Formazan extraction (Day 3)(a) Prior to extraction, label 1.5 ml conic “safelock” type micro tubes with the chemicals code number
and replicate N° (1 tube/epidermis).
Note: Steps (b) to (d) should be followed chemical by chemical.
(b) Place the 3 tissue units on absorbent paper to dry the tissues.
(c) After placing the epidermis units on the plate lid (as a support), make a total biopsy of the epidermis byusing the special biopsy punch.
(d) Gently separate the epidermis from the collagen matrix with the aid of forceps, and place both parts(epidermis and collagen matrix) into the labeled microtubes.
Important:- Place the epidermis treated side onto the collagen matrix.
- If the collagen matrix is colored by the chemical, replace it during the extraction by a
new collagen matrix (biopsy punched) taken from killed de-epidermised non treatedepidermis.
(e) When all the tissues of an application set are punched and ready in the micro tubes for, add 500 µl of
acidic isopropanol per tube.
(f) Plug each tube to avoid evaporation and mix thoroughly using a vortex mixer.
(g) Ensure that all the biological material is correctly immersed in the solvent.
Record start time of extraction in MDS Annex 9.
(h) For readings on the same day as extraction:
Store 4 hours at room temperature (recommended +18 to 23°C) protected from light.
Vortex each tube at the middle of the incubation period to help extraction.
or
For readings on Monday morning: store refrigerated at +4°C protected from light.
Record timings and necessary details in MDS Annex 9.
(i) Remove (using suction pump and toxic waste trap) the MTT solution from each well of the remaining
plate. Discard the empty plate in the special waste container.
At the end of the formazan extraction period :(j) Mix each tube using a vortex mixer, until solution color becomes homogeneous.
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© ECVAM DB-ALM: Protocol(k) If any visible cell/tissue fragments are in suspension, centrifuge (500 rpm) to eliminate the fragments
and avoid further possible interference with the absorbance readings.
Absorbance / Optical Density measurements (Day 3 or 6)(a) Transfer per tissue 2 x 200 µL sample/well (=2 wells per tissue) from each tube into a 96 wellflat bottom microtiter plate (labeledappropriately).
Note: Beware of isopropanol evaporation in 96
well plates. It is recommended to fill not morethan 42 wells per plate and to make all readingsin the same run (ex: maximum area to be used ismarked in green).

(b) Read the Optical Densities (OD) in a 96-wellplate spectrophotometer using wavelength filter
centered at 570 nm pass-band ± 30 nm andacidified isopropanol solution as blank without
using a reference filter.
Note: Readings are performed without reference
filter, since the "classical" reference filter often
used in the MTT test (630 nm) is still within theabsorption curve of formazan. Since filters havea tolerance their use can lead to reduction of the
dynamics of the signal (OD).

Do not use the “empty” wells for readings (see example below).

(c) Record results on prepared templates. Templates are normally linked to specific readers/programs
used by the test laboratory and may slightly differ form the example above. A direct print of the readings
should be made immediately and identified with date and signature and kept as raw data.
Acceptance Criteria
EpiSkinT M -SM kits are manufactured according to defined quality assurance procedures (certified ISO9001). All biological components of the epidermis and the kit culture medium have been tested for theabsence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by an MTT
cytotoxicity test using sodium dodecyl sulphate (SDS) and by histological scoring. Additional criteria
could be added in the future.
Method Documentation Sheets (MDS) allow for compliant Quality Control: correct set up, calibration,function of the equipment and quality of the preparations are an intergal part of the protcol. See
Annexes 1 to 13.
Assay acceptance criteria for negative and positive controls
Negative control (NC) acceptance: OD of the negative control (PBS treated) reflects theviability of the tissues used in the test conditions (after shipping, storing in specificconditions). An absolute OD below the historical established lower boundary of theconfidence interval indicates abnormal viability of tissues thus indicating possible
difference in sensitivity to chemicals. These tissues should not be used for this application.
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It can be considered that the NC meets the acceptance, if the mean OD value of the 3 tissues is> 0.6 and the Standard Deviation value (SD) of the % viability is 18.
Positive control (PC) acceptance: OD of the positive control (5% aq. SDS-treated) reflects
the sensitivity of the tissues used in the test conditions (after shipping, storing in specificconditions). One positive control should be included in each run (maximum one PC perday).
It can be considered that the Positive Control meets the acceptance if the mean viability
expressed as % of the NC, is 40% and the SD is ≤ 18.
Batch acceptance: all chemical data from one batch are accepted if both the negative and the positive
control fulfill the above requirements.
Chemical data acceptance: The inter-batch viability mean is calculated from the three independent
assays using intra-batch tissue mean. The 3 intra-batch means must predict the same class of irritation.
For a given chemical, if the batch gives an SD > 18, the chemical is retested once (possible technicalproblem or error). If 2 or 3 batches give SDs > 18 the assay is not repeated (variability probably linked
to the chemical itself).

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Data Analysis
All data/calculations should be recorded on prepared Data Report Forms. Excel prepared spreadsheets
could be used (not provided by EpiSkin supplier). Blank data and chemicals data (OD) are copied andpasted in the Excel prepared tables named: “IMPORT sheet”.
Note: The figures below are snapshots of the actual calculation spreadsheets and are presented here forillustration only.

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Data calculation steps
These calculation steps are applicable to the majority of test-substances characterized as follows: nointeraction with the MTT reagent, non colored, with a low ability to stain the tissues and measured non
specific color value 5% relative to negative control.
Main steps to be followed by the prepared template(a) Blanks: calculate the OD mean from the 6 replicates for each plate.
(b) Negative PBS-treated controls: Subtract blanks mean value from individual tissues OD. Corrected OD
mean for the 3 tissues corresponds to 100% viability.
(c) Positive control (SDS 5%): Subtract blanks mean value from individual tissues OD. Calculate the OD
mean for each individual tissue.
(d) Tested compound: Subtract blanks mean value from individual tissues OD. Calculate the OD meanfor each individual tissue.
(e) The viability % for each treated epidermis, are calculated relative to the mean of negative controls.
(See formula below)(f) Standard deviations are calculated on OD and % viabilities.
(g) CV are calculated on % viabilities.
The mean OD of the three Negative Controls (PBS treated) corresponds to 100% reference
viability.
For each individual treated tissue (TT) with a test substance or the positive control (PC), therelative viabilities are calculated as follows:
OD Treated tissue
OD Negative control (ODNC)OD Positive control (ODPC)= OD TTraw - OD blank mean
= OD NCraw - OD blank mean
= OD PCraw - OD blank mean

Individual viabilities (%)%Positive Control1 = [OD PC1 /mean OD NC ] x 100%Positive Control2 = [OD PC2 /mean OD NC ] x 100

The mean relative viability is used for classification according to the prediction model.
Data calculation for MTT interacting substances
Chemicals that interfere with MTT can produce non specific reduction of the MTT. It is necessary toevaluate the OD due to the non specific reduction and to subtract it before calculations of viability %.
Non specific MTT reduction calculation (NSMTT):
ODku: untreated killed tissues
ODkt: chemical treated killed tissues
NSMTT= [(ODkt - ODku ) / ODNC] x 100
Note: NSMTT should be ≤ 50% relative to the negative control OD.
True MTT metabolic conversion (TODTT):ODTV: chemical treated viable tissues
TOD TT: true MTT metabolic conversion for treated tissue
TOD TT = [ODTV – (ODkt - ODku )]Relative viability% tissue1 = [TODTT1 / OD NC ] x 100
Relative viability% tissue2 = [TODTT2 / OD NC ] x 100
Relative viability% tissue3 = [TODTT3 / OD NC ] x 100

Remark: If non specific MTT reduction is > 50% relative to the negative control (living epidermis),
additional steps must be undertaken if possible or the chemical must be considered as incompatible
with the test.
Data calculation for dyes and coloring test substances able to stain tissues
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For chemicals detected as able to color the tissues, it is necessary to evaluate the non specific OD due tothe residual chemical color (unrelated to mitochondrial activity) and to subtract it before calculations of
the “true” viability %.
Non specific color % (NSC%):ODCT: chemical treated tissue (not incubated with MTT)ODNC: Control PBS treated tissue (incubated with MTT)
NSC%= [ODCT / OD NC ] x 100
Note: NSC should be: 5% < NSC% ≤ 50%. If NSC% is < 5% then use normal calculation mode.
True MTT metabolic conversion (TODTT):ODTV: chemical treated viable tissues
TOD TT: true MTT metabolic conversion for treated tissue
TOD TT = [ODTV – ODCT]
Relative viability% tissue1 = [TODTT1 / OD NC ] x 100
Relative viability% tissue2 = [TODTT2 / OD NC ] x 100
Relative viability% tissue3 = [TODTT3 / OD NC ] x 100
Remark: If non specific OD due to coloration is > 50% relative to the negative control, additional steps
must be undertaken if possible or the chemical must be considered as incompatible with the test.
Prediction Model
Irritation potential of test chemical is determined according to the UN GHS and EU CLP as "Category 2:
Irritant" or “No Category” (UN, 2009; No. 1272/2008, EU, 2008). An irritant 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.
The prediction model is defined as described below:
In vitro results

In vivo classification

Mean tissue cell viability ≤ 50%

Category 2: Irritant

Mean tissue cell viability > 50%

No Category

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Annexes
Pages below contain snapshots from the validation study SOP. The actual MDS sheets are attached to theprotocol and can be downloaded and printed separately, if needed. Go to DB-ALM webite(http://ecvam-dbalm.jrc.ec.europa.eu/), section related to protocol No. 131 and select Related
information: Downloads.

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