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IN VITRO SKIN IRRITATION TEST:HUMAN EPIDERMIS MODEL
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Japan Tissue Engineering Co., Ltd.

-JSAAE Skin Irritation Test Validation Study-

SKIN IRRITATION TESTUSING THE RECONSTRUCTED HUMAN MODEL“LABCYTE EPI-MODEL 24”
Ver. 8.3

LabCyte EPI-MODEL24 SKIN IRRITATION TEST-42 HOURS

S.O.P.

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TABLE OF CONTENTS
1.

RATIONAL AND BACKGROUND................................................................................................... 4
1.1
LabCyte EPI-MODEL 24 SKIN IRRITATION TEST (SIT using LabCyte EPI-MODEL24)........ 4
1.2
BACKGROUND OF SIT using LabCyte EPI-MODEL24 .......................................................... 4
1.3
BASIS OF THE METOD........................................................................................................... 4
1.3.1
1.3.2

1.4
1.5
1.6
2.

2.2
2.3
2.4
2.4.1
2.4.2

LabCyte EPI-MODEL 24 KIT COMPONENTS .............................................................................7
SHIPMENT OF LabCyte EPI-MODEL24......................................................................................7
INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL 24............................................................7

TEST CHEMICALS .................................................................................................................. 7
CONSUMABLES...................................................................................................................... 7
OTHERS................................................................................................................................... 8
EQUIPMENT / INSTRUMENTS ...................................................................................................8
CONSUMMABLE ITEMS .............................................................................................................8

TEST METHOD ............................................................................................................................... 9
3.1
PREPARATIONS...................................................................................................................... 9
3.1.1
3.1.2
3.1.3
3.1.4

3.2
3.2.1
3.2.2

3.3
3.3.1
3.3.2
3.3.3
3.3.4
3.3.5

4.

LIMITATION OF THE METHOD............................................................................................... 6
BRIEF BASIC PROCEDURE................................................................................................... 6
DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)......................................... 6

MATERIALS .................................................................................................................................... 7
2.1
LabCyte EPI-MODEL 24 .......................................................................................................... 7
2.1.1
2.1.2
2.1.3

3.

TEST SYSTEM DESCRIPTION...................................................................................................4
ASSAY QUALITY CONTROL.......................................................................................................5

MTT SOLUTION ..........................................................................................................................9POSITIVE CONTROL SUBSTANCE............................................................................................9
NEGATIVE CONTROL SUBSTANCE ..........................................................................................9POLY WASH BOTTLE FOR PBS.................................................................................................9

TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT............ 10
DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE ...............................................10
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTT .............................................. 11

EXECUTION OF THE TEST .................................................................................................. 13
PREPARATION OF LabCyte EPI-MODEL 24 (DAY -1)..............................................................13
APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0) ................................................13
POST TREATMENT INCUBATION (DAY 0~2)...........................................................................16
MTT ASSAY (DAY 2) ..................................................................................................................16
FORMAZAN EXTRACTION AND MEASUREMENT (DAY 2~3) ................................................17

ASSESSMENT .............................................................................................................................. 19
4.1
CONDITIONS FOR A SUCCESSFUL STUDY....................................................................... 19
4.2
ASSAY CRITERIA .................................................................................................................. 19

MDS 1: RECEIPT OF LABCYTE EPI-MODEL 24 ............................................................................. 21
MDS 2: PRE-INCUBATION OF LABCYTE EPI-MODEL 24 (Section 3.3.1) .................................... 22
MDS 3-1: APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section
3.1.2, 3.3.2 ~ 3.3.3) ............................................................................................................................... 23
MDS 3-2: APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section
3.3.2~3.3.3) ........................................................................................................................................... 24
MDS 4: MTT ASSAY (Section 3.3.4) ................................................................................................. 25
MDS 5: FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.5) .................................. 26

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REVISION HISTORY............................................................................................................................. 27

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1. RATIONAL AND BACKGROUND
1.1

SKIN IRRITATION TEST using LabCyte EPI-MODEL 24 (SIT using LabCyte EPI-MODEL24)
The SIT using LabCyte EPI-MODEL24 is designed for the prediction of acute skin irritation ofchemicals by measurement of its cytotoxic effect, as reflected in the MTT assay, on the
Reconstructed Human Epidermis (RHE) model. The SIT using LabCyte EPI-MODEL24 is not a kit;
LabCyte EPI-MODEL24 tissues are commercially available per tissues item (with a minimum of 24
LabCyte EPI-MODEL24 tissues per order).

1.2 BACKGROUND OF SIT using LabCyte EPI-MODEL24
Performance standards for applying human skin models to in vitro skin irritation testing werealso defined based on the validated test EpiSkinTM test method (ECVAM SIVS, 2007). These
performance standards can be then used to evaluate the accuracy and reliability of otheranalogous test methods (also referred to as “me-too” tests) that are based on similar scientificprinciples and measure or predict the same biological or toxic effect.
Based on the GHS-EU classification, 12 irritants and 13 non-irritants in the draft performancestandards (ECVAM 2007) and the statement by ESAC (ESAC2009) were performed the validation
study through the 7 labs SIT using LabCyte EPI-MODEL24. Results were summarized at JSAAE
1st report and 2nd report on this validation study.

1.3 BASIS OF THE METHOD
Chemical-induced skin irritation, manifested by erythema and oedema, is the results of acascade of events beginning with penetration of the stratum corneum and damage to theunderlying layers of keratinocytes. The dying keratinocytes release mediators that begin the
inflammatory cascade which acts on the cells in the dermis, particularly the stromal andendothelial cells. It is the dilation and increased permeability of the endothelial cells that produce
the observed erythema and oedema. The RhE-based test methods measure the initiating eventsin the cascade.
The relative viability of the treated tissues was measured at the end of the treatment exposure(15 minutes) followed by a post-exposure period (42 hours) using MTT [(3-4,5-dimethyl thiazole2-yl) 2,5-diphenyltetrazoliumbromide] assay. A cutoff value of 50% viability of the negative control
value was considered and used to classify test substances as irritant (I) or non irritant (NI). The
culture environment might allow the detection of very small quantities of cytokines secreted by the
epidermis in response to topical application of test substances.

1.3.1
TEST SYSTEM DESCRIPTION
LabCyte EPI-MODEL24 is a new, commercially available RhE model produced by Japan Tissue
Engineering Co. Ltd. It consists of normal human epidermal keratinocytes whose biological origin
is neonate foreskin. In order to expand human keratinocytes while maintaining their phenotype,they were cultured with 3T3-J2 cells as a feeder layer (Rheinwald and Green, 1975; Green, 1978).
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Reconstruction of human cultured epidermis is achieved by cultivating and proliferating
keratinocytes on an inert filter substrate (surface 0.3 cm2) at the air-liquid interface for 13 days with
an optimized medium containing 5% fetal bovine serum. It constructs a multilayer structure
consisting of a fully differentiated epithelium with features of the normal human epidermis,
including a stratum corneum. LabCyte EPI-MODEL24 is embedded in an agarose gel containing
nutrient solution and shipped in 24-well plates at around 18°C.
1.3.1.1 Quality control of the test system
The LabCyte EPI-MODEL24 is manufactured according to defined quality assurance
procedures. Each batch production was provided with quality controls such as storage conditions,
RHE instructions for use, lot number and origin, histology (demonstration of human epidermis-likestructure with multilayered stratum corneum), cell viability, barrier function integrity (0.14 ≤ IC50 ≤0.4).

1.3.1.2 Precautions
The epidermal cells are taken from healthy donor negative to HIV, and Hepatitis. Nevertheless,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 special containers or autoclaving).

1.3.2

ASSAY QUALITY CONTROL

1.3.2.1 Assay Acceptance Criterion 1: Negative Control
The absolute OD of the negative control (NC) tissues (treated with sterile DPBS) in the MTT
assay is an indicator of tissue viability obtained in the testing laboratory after shipping and storingprocedures and under specific conditions of use.
0.7 ≤ Mean OD (A570/650) measured value ≤ 2.5
1.3.2.2 Assay Acceptance Criterion 2: Positive Control
A 5% SDS (in H2O) solution (see 7.6.3) is used as positive control (PC) and tested concurrently
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±1 %
confidence interval of the historical data.
Mean tissue viability ≤ 40%
1.3.2.3 Assay Acceptance Criterion 3: Standard Deviation (SD)
Since in each test skin irritancy potential is predicted from the mean viability determined on 3
single tissues, the variability of tissue replicates should be acceptably low.
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Standard Deviation (SD) of tissue viability of 3 identically treated replicates for negative
control and positive control ≤ 18 %1.4 LIMITATION OF THE METHOD
One limitation of this assay method is a possible interference of the test substance with the MTT
endpoint. A colored test substance or one that directly reduces MTT (and thereby mimics
dehydrogenase activity of the cellular mitochondria) may interfere with the MTT endpoint. However,these test substance are a problem only if at the time of the MTT test (i.e. 42 hours after test
substance exposure) sufficient amounts of the test substance are still present on (or in) the tissues.
In case of this unlikely event, the (true) metabolic MTT reduction and the contribution by a colored
test material or (false) direct MTT reduction by the test material can be quantified by a proceduredescribed in Section 3.2.
The method is not designed for testing of highly volatile test substances, gases and aerosols.

1.5 BRIEF BASIC PROCEDURE
On the day of receipt, LabCyte EPI-MODEL24 tissues are conditioned by incubation to releasetransportstress related compounds and debris overnight. After pre-incubation, tissues are topically
exposed to the test chemicals for 15 minutes. Preferably, three tissues are used per test chemical(TC) and for the positive control (PC) and negative control (NC). Tissues are then thoroughly
rinsed, blotted to remove the test substances, and transferred to fresh medium. After 42 hr
incubation period, the MTT assay is performed by transferring the tissues to the well containing
MTT medium (0.5 mg/ml). After 3 hr MTT incubation, the blue formazan salt formed by cellular
mitochondria is extracted with 0.3 mL/tissue of isopropanol and the optical density of the extracted
formazan is determined using a spectrophotometer at 570 nm and 650 nm as reference. Relativecell viability is calculated for each tissue as % of the mean of the negative control tissues. Skin
irritation potential of the test material is predicted if the remaining relative cell viability is below
50%.

1.6 DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)
According to the GHS classification (Category 2 or no label), 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.
In vitro results

In vivo prediction

Tissue viability is ≤ 50%

Irritant

Tissue viability is > 50%

Non Irritant

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2. MATERIALS
2.1

LabCyte EPI-MODEL 24

2.1.1
LabCyte EPI-MODEL 24 KIT COMPONENTS
LabCyte EPI-MODEL 24 kit components are shown in Table 1.
Table 1 - LabCyte EPI-MODEL24 Kit Components
Component
Qty
LabCyte EPI-MODEL 24 plate

Assay Medium
24-well plate

2.1.2

Description

1 plateContains 24 culture inserts with tissues fixed innutritive agar medium for transport (usable area:
0.3cm2).

1 bottle

Basic medium for incubation (30mL). Store at
refrigeration temperature.

1 plateBlank plate forroom-temperature.

use

in

assay.

Storeat

SHIPMENT OF LabCyte EPI-MODEL24

LabCyte EPI-MODEL 24 is packed in a special container (Icompo/NIPPON EXPRESS CO.,LTD) and delivered by NIPPON EXPRESS CO., LTD. After the Icompo is delivered, examine thecontents and make sure that all kit components (LabCyte EPI-MODEL24 plate, assay medium,and 24-well assay plate) are included in the package. Confirm lot numbers and expiration dates
also. Record details in the Methods Documentation Sheet (MDS) 1.
NIPPON EXPRESS will pick up the Icompo at a later date (generally, the day after the date ofdelivery), and we ask that you return it with a slip documenting receipt, as well as the insulating
materials.

2.1.3

INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL 24

Begin incubating all of the culture inserts after opening the package. Do not store the culture
inserts again after opening.
The human epidermis cells used in LabCyte EPI-MODEL 24 originate from a normal donor andare HIV-, HBV-, HCV-, and HPV-negative. However, handle them with enough care and inaccordance with the laboratory biosafety guidelines since they contain raw materials of humanorigin.

2.2 TEST CHEMICALS
Coded test chemicals are delivered to each laboratory.

2.3

CONSUMABLES
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The following consumables are required.
* The described quantities are necessary so that 1 to 6 samples can be assayed once.
• Assay Medium, 100mL (J-TEC: 402250)
1 bottle• MTT, 25mg (J-TEC: 403026)
1 bottle• Wide orifice cell saver tips for micro-pipettes (sterile) 96 tips
1 box
• 24-well assay plate (Becton,Dickinson and Company: 353047)
7 plates• 96-well plate (Becton,Dickinson and Company: 353072)
1 plates• Phosphate buffered saline (PBS) 500mL (Invitrogen: 14190-144)
2 bottles• Isopropanol 500mL (Wako Pure Chemical Industries: 164-08335)
1 bottle• SLS 25g (SIGMA:L4390)
1 bottle• Sterile distilled water 20mL (Otsuka Pharmaceutical: 36A1X00001) 1 bottles• Sterile cotton buds (JAPAN COTTON BUDS: 10A754D)
1 box

2.4

OTHERS

2.4.1

EQUIPMENT / INSTRUMENTS
•
•
•
•
•
•
•
•
•
•
•
•
•

2.4.2

Safety cabinet (or clean bench)
Water bath (37 °C)
CO2 incubator (37 °C, 5%CO2, capable of maintaining high humidity)
Autoclave
96-well multi-plate reader (required filters: 450nm, 570nm, 650nm)
Precision balance (0.1mg)
Aspirator
Stop-watches
Adjustable micro-pipette (10-200µL, 200-1000µL)
Sharp-edged forceps (sterile)
Micro spatula (sterile)
Beaker (1~2L: sterile)
Sterilizable poly wash bottle (500~1000mL: sterile)

CONSUMMABLE ITEMS•
Micro-pipette tips (sterile: 10~200µL, 200~1000µL)•
Microtubes (1.5mL)•
Scalpel (KEISEI MEDICAL INDUSTRIAL: Keisei Scalpel 11A)

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3. TEST METHOD*Perform operations in Section 3.1.1~3.1.4 and Section 3.3.1~3.3.2 aseptically in a safetycabinet (or clean bench).
*Operations other than above do not need to be performed with an aseptic technique. For
these operations, refer to Section 2.1.3 INSTRUCTIONS FOR USE OF LabCyte
EPI-MODEL 24

3.1

PREPARATIONS

3.1.1
MTT SOLUTION(1) Dissolve MTT in the assay medium to prepare the MTT medium (final concentration:
0.5mg/mL)
Use ultrasonic cleaning equipment or a vortex mixer as necessary in order to completely
dissolve the MTT.
*Store in a dark, cold place and use it within 24 hours.
(2) Record details of step (1) above in the MDS 4.

3.1.2
POSITIVE CONTROL SUBSTANCE(1) Weigh 500mg of SLS precisely.
(2) To prepare a positive control solution, put the SLS into a graduated cylinder or measuring
flask and dilute to 10mL with distilled water (final concentration: 5% w/v)]* Store in a dark, cold place and use it within 24 hours.
(3) Record details of steps (1) and (2) above in the MDS 3.

3.1.3
NEGATIVE CONTROL SUBSTANCE(1) Use distilled water.

3.1.4
POLY WASH BOTTLE FOR PBS(1) Sterilize poly wash bottle using an autoclave.
(2) Fill the sterilized poly wash bottle aseptically with sterile PBS.

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TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT

There are two kinds of test chemicals that interfere with the MTT assay as follows.
(a) Chemical that stains epidermis tissues.
(b) Chemical that is able to directly reduce MTT.
Test chemical that stains the epidermis tissues has a possibility to transfer from the epidermis
tissues to the extraction solution and to affect the optical density (OD) measurements.
Test chemical that is able to directly reduce MTT can affect the optical density (OD)measurements, if the test chemical is present in the epidermis tissues when the MTT viability
test is performed. Detection procedure of these test chemicals is described below.

3.2.1
3.2.1.1

DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE
STEP1 (PRELIMINARY TEST)(1) Add 25µL (Liquid) or 25mg (Solid) of the test chemical into wells of 24-well assay plate
preliminarily filled with 0.5mL of distilled water. Untreated distilled water is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 15
minutes.
(3) After incubation, shake the mixture gently and evaluate the staining of the distilled water
macroscopically.
(4) When the color of the solution changes significantly, the test chemical is presumed to havethe potential to stain the tissue and a functional check on viable tissues (Step2) should be
performed. When the color of the solution does not change significantly, the test chemical isdetermined not to have a potential to stain the tissue.
3.2.1.2

STEP2 (FUNCTIONAL CHECK ON VIABLE TISSUE)(1) Add 25µL (Liquid) or 25mg (Solid) of the test chemical, which clearly changed the color ofthe distilled water (Step1), onto the surface of the epidermis tissues. Distilled water is usedas negative control.
(2) Follow all procedures described in this SOP Section 3.3 EXECUTION OF THE TEST.
However, incubate the tissue for 3 hours in culture media without MTT instead of incubating
in media containing MTT to evaluate the staining of the epidermis tissues.
(3) Calculate ratio of staining by test chemical from the following formula.
Ratio of staining by test
chemical (%) =OD test chemical – OD negative control
OD negative control

×100(4) When the ratio of staining by test chemical is <5%, correction of the results is not necessary.
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When the ratio is between 5% and 30%, the corrected MTT OD is calculated using thefollowing formula.test chemical
Corrected MTT OD =OD stained tissue (MTT assay)-OD stained tissue (no MTT assay)

When the ratio of staining by test chemical is >30%, the test chemical must be considered
incompatible with the test. However, when the Cell viability (%), which is calculated
according to the procedures described in this SOP Section 3.3.5.2, is <50%, the test
chemical is determined as irritant. Therefore correction of the results or determination ofincompatibility of the test chemical is not necessary.

3.2.2
3.2.2.1

DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTTSTEP3 (PRELIMINAY TEST)(1) Add 25µL (Liquid) or 25mg (Solid) of the test chemical into wells of 24-well assay plate
preliminarily filled with 0.5mL of MTT medium. Untreated MTT medium is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 1 hour.
(3) After incubation, shake the mixture gently and evaluate the staining of the MTT medium
macroscopically.
(4) When the MTT medium turns blue/purple significantly, the test chemical can reduce MTT
and additional functional check (Step4) must be performed.

5% SLS

1-bromo hexane

1,1,1-tetrachloroe

5-(1,1-dimethylet

thanehyl)-2-methyl

cinnamaldehyde

eugenol

Photo 1 – Example of test for direct MTT reduction ability (STEP 3). Test substances5-(1,1-dimethylethyl)-2-methyl, cinnamaldehyde and eugenol and have directlyreduced MTT. In these cases, Step 4 must be performed.

3.2.2.2

STEP4 (FUNCTIONAL CHECK ON VIABLE TISSUE)(1) Add 25µL (Liquid) or 25mg (Solid) of the test chemical, which clearly changed the color of
the MTT medium into blue/purple (Step3), onto the surface of the epidermis tissues.
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Distilled water is used as negative control.
(2) Follow all procedures described in this SOP Section 3.3 EXECUTION OF THE TEST.
However, use the epidermis tissues that has been freeze-killed at -20 °C or lower for morethan 24 hours instead of viable epidermis tissues.
(3) Calculate ratio of staining by test chemical from the following formula.
Ratio of staining by test
chemical (%) =OD test chemical – OD negative control
OD negative control

×100(4) When the ratio of staining by test chemical is <30%, correct OD data using the following
formula.

Corrected OD =OD (viable tissue) test chemical – [OD (freeze-killed tissue) test chemical- OD (freeze-killed tissue) negative control]

When the ratio of staining by test chemical is >30%, the test chemical must be considered
incompatible with the test. However, When the Cell viability (%), which is calculated
according to the procedures described in this SOP Section 3.3.5.2, is <50%, the test
chemical is determined as irritant. Therefore correction of the results or determination ofincompatibility of the test chemical is not necessary.

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EXECUTION OF THE TEST

3.3.1 PREPARATION OF LabCyte EPI-MODEL 24 (DAY -1)(1) Pre-warm the assay medium for 30 minutes to
Figure 1
Assay Plate
37 °C using a water bath.
(2) Fill 3 wells of the 1st row of each 24-well assay 1 row(substance application)plate with the pre-warmed assay medium
2 row(0.5mL/well).
3 row→ Figure 1(post-incubation)(3) Open the LabCyte EPI-MODEL 24 aluminum
4 row(MTT assay)
package.
(4) Open the LabCyte EPI-MODEL 24 plate lid
and pick up the culture inserts using sterile forceps.
*Do not touch the epidermis surface of culture inserts.
*Use forceps to remove agar medium sticking to the outside of the culture inserts.
(5) Transfer the culture inserts into assay
Figure 2
medium filled wells of the 1st row using sterile
Assay Plate
forceps.
1 row→ Figure 2(substance application)*Avoid air bubble formation under the tissue 2 row
inserts.
3 row(post-incubation)(6) Place the plate (lid on) in a CO2 incubator.
row(7) Incubate overnight (15~30 hours) until 4(MTTassay)
Section 3.2.2 “APPLICATION OF TESTCHEMICALS AND RINSING.”(8) Record details of steps (1) - (7) above in the MDS 2.
st

nd

rd

th

st

nd

rd

th

3.3.2

APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0)

3.3.2.1 PREPARATION OF WELLS FOR POST-INCUBATION (3RD ROW)
(1) Pre-warm the assay medium for 30 minutes to 37 °C using a water bath.
(2) Remove the assay plate from the CO2
Figure 3
incubator.
(3) Open the lid of the assay plate, and fill 3 wellsof the 3rd row with the pre-warmed assay
medium (1.0mL/well) using a micropipette.
→ Figure 3(4) Place the plate (lid on) in a CO2 incubator.

2 row(5) Incubate until application of test chemicals

4 row

Assay Plate

st

1 row
(substance application)
nd

rd

3 row
(post-incubation)th(MTT assay)(0~12 hours).
(6) Record details of steps (1) – (5) above in the MDS 3.

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APPLICATION OF TEST CHEMICALS(1) Remove the assay plate from the CO2 incubator.
(2) Apply test chemicals onto the surface of epidermis tissues in the 1st row of the assay plate.
Use 3 wells per test chemical (N=3).
FOR LIQUIDs:

Carefully apply 25µL of the test chemical onto the central part of each

epidermis using a micropipette.

After applied, close the lid of the assay plate and tap the sideof the plate outside the safe cabinet (or clean bench) in order for the liquid to spread out overthe entire epidermis surface.

If necessary, use a

Photo 2 - Pipette tips for viscous liquids

micro spatula to coat the unapplied surface withliquids.

Do not push the epidermis surface too

hard with the spatula.
*Use wide orifice cell saver tips for viscous liquids.
→ Photo 2
Use a pipette, etc. to familiarize yourself with thenature of the test chemicals in advance.
FOR SOLIDs:

Weigh out 25mg (±1mg) of the solid
Photo 3 – Applying a solid substancechemical with a precision balance in advance.
Apply first 25µL of distilled water and then theweighed test chemical onto the epidermis surface.
Use a micro spatula if necessary to gently spreadthe test chemical.
→ Photo 3*One 24-well assay plate should be used to assay
only one test chemical.
→ Figure 4

Figure 4(1 samples x 3(n) = 3 (culture inserts))

Assay Plate
Test substance(3) Apply test chemical onto each well at

st

1 row
(substance application)

1~3-minute intervals.

nd

2 row

(4) Incubate each well for 15 minutes in therdcabinet (lid on between the intervals).
*Close the lid of the assay plate at all times
except when applying samples.

3 row(post-incubation)
th

4 row
(MTT assay)

It might

affect the amount of test sample if the lid is kept open, due to air circulation in the safe
cabinet (or clean bench).
(5) Record details of steps (1) - (4) above in the MDS 3.

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3.3.2.3 REMOVAL OF THE TEST CHEMICALS(1) 15 minutes (±30 seconds) after applying a chemical, open the assay plate and pick up a
culture insert with sterile forceps. Discard test

Photo 4 - Rinse 1

chemicals on the tissue by tilting and then tapping
the insert on the beaker.
(2) Fill and overflow the culture insert with PBS using a
PBS filled poly wash bottle. Hit the PBS stream
from the washing bottle on the side-wall of theculture insert and wash on the tissue surface by the
PBS current.
→ Photo 4
Attention: Must not to hit the PBS stream on the

Photo 5 - Rinse 2

tissue surface directly. Be careful not todamage the tissue surface.
(3) Discard the PBS into a beaker by tilting the insert.
If necessary, remove the PBS inside the culture
insert by tapping it on the beaker only once.
→ Photo 5(4) Repeat steps (2) and (3) at least 15 times or moreas much as possible, and remove all residual test

Photo 6 - Rinse 3

chemical on the tissue surface almost completely.
Must not do discarding by tapping at only the lastwashing operation.
(5) Gently remove the leftover PBS outside the culture
insert with a sterile cotton bud. But don’t touch
inside the culture insert by a cotton bud.
→ Photo 6
Attention: Even if residues of washing PBS remain on the tissue surface, don’t do at all
because it is not necessary to
remove them.

Test substance(6) If test material remains on the epidermis
stsurface, repeat steps (2) ~ (5) again.

1 row(substance application)(7) Transfer the blotted culture insert to a well in
the

3rd

row

of

post-incubation).
→ Figure 5

theAssay Plate

Figure 5samecolumn

(for

nd

2 row

rd

3 row
(post-incubation)
th

4 row
(MTT assay)

15

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*Avoid air bubble formation under the culture inserts.
(8) Repeat steps (1) ~ (7) for all the culture inserts at 1~3-minute intervals.
(9) Record details of steps (1) – (8) above in the MDS 3.

3.3.3

POST TREATMENT INCUBATION (DAY 0~2)

(1) Close the lid of the assay plate and place it in a CO2 incubator.
(2) Incubate for 42 hours.

3.3.4

MTT ASSAY (DAY 2)

3.3.4.1

PREPARATION OF WELLS FOR MTT ASSAY(1) Pre-warm MTT medium for 30 minutes to 37°C using a water bath.
(2) Remove the assay plate from the CO2 incubator.
Assay Plate

Figure 6(3) Open the lid of the assay plate, and fill each well

Test substanceof the 4th row with the pre-warmed MTT medium

st

1 row
(substance application)(0.5mL/well) using a micropipette.

nd

2 row

→ Figure 6
rd

3 row

(4) Close the lid of the assay plate and place it in the(post-incubation)

CO2 incubator.

th

4 row
(MTT assay)(5) Incubate until starting MTT assay (about 0 ~ 12hours).
(6) Record details of steps (1) – (5) above in the MDS 4.

3.3.4.2

MTT ASSAY(1) Remove the assay plate from the CO2 incubator after 42 hours (±1 hour) of post-incubation.
(2) Transfer each culture insert from the 3rd row to
the 4th row of the corresponding column.
→ Figure 7*Avoid dripping from the base end surface of

Figure 7

Assay Plate
Test substance

st

1 row
(substance application)
nd

2 row

the culture insert into other wells.
rd*Avoid air bubble formation under the culture
inserts.

3 row(post-incubation)
th

4 row
(MTT assay)(3) Close the lid of the assay plate and place it in
the CO2 incubator.
(4) Incubate for 3 hours.
16Version 8.3

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(5) Record details of steps (1) – (4) above in the MDS 4.

3.3.5

FORMAZAN EXTRACTION AND MEASUREMENT (DAY 2~3)

3.3.5.1

FORMAZAN EXTRACTION(1) Remove the assay plate(s) from the CO2 incubator 3 hours (±5 minutes) after the MTT
assay.
(2) Open the lid of the assay plate and pinch the cultured epidermis from each culture insert of
the 4th row with forceps.

Photo 7 - Detachment of epidermis→ Photo 7*Use a micro spatula to scratch up the epidermis
or a scalpel to cut the membrane filter on the baseof the culture insert if the cultured epidermis
cannot be pinched due to damage from a test
chemical.
(3) Transfer the epidermis tissue into a 1.5mL microtube.
(4) Add 300µL of isopropanol to the micro tubes andsoak the entire epidermis tissue in the isopropanol.

(5) Incubate the micro tubes in a dark cold place (or refrigerator) overnight (more than 15hours) in order to completely extract pigments.
*Tighten the micro tube seal.
*Periodically shaking the micro tubes will contribute to a more
efficient extraction.
(6) Shake the micro tubes to mix the solution.
*If split epidermis tissues are suspended, wait until they sink or
gently centrifuge them (if a centrifuge is available).
(7) Transfer 200µL of the solution in each micro tube into each well on a 96-well plate.
*One well of 200µL of isopropanol should be set as a blank.
*Figure 8 shows an example of allocation in a 96-well plate.
Figure 8 – Allocation for a 96-well plate
A

1
Blank

B

DW-1

C

DW-2

D

DW-3

E
F
G

5%
SLS-1
5%
SLS-2
5%
SLS-3

2

3

4

5

6

7

8

9

10

11

Sample1-1
Sample1-2
Sample1-3
Sample2-1
Sample2-2
Sample2-3

Sample3-1
Sample3-2
Sample3-3Sample4-1
Sample4-2
Sample4-3

Sample5-1
Sample5-2
Sample5-3
Sample6-1
Sample6-2
Sample6-3

Sample7-1
Sample7-2
Sample7-3
Sample8-1
Sample8-2
Sample8-3

Sample9-1
Sample9-2
Sample9-3
Sample10-1
Sample10-2
Sample10-3

Sample11-1
Sample11-2
Sample11-3
Sample12-1
Sample12-2
Sample12-3

Sample13-1
Sample13-2
Sample13-3
Sample14-1
Sample14-2
Sample14-3

Sample15-1
Sample15-2
Sample15-3
Sample16-1
Sample16-2
Sample16-3

Sample17-1
Sample17-2
Sample17-3
Sample18-1
Sample18-2
Sample18-3

Sample19-1
Sample19-2
Sample19-3
Sample20-1
Sample20-2
Sample
20-3

12

H

17

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(8) Record details of steps (1) – (7) above in the MDS 5.

3.3.5.2

OPTICAL DENSITY MEASUREMENTS OF THE EXTRACTS(1) Using a 96-well plate reader, measure optical densities (OD) at 570nm and 650nm anddetermine the measured OD by subtracting the 570nm OD from the 650nm OD.
The equation is shown below:
Measured OD = [570nm ODsample – 570nm ODblank] – [650nm ODsample – 650nm ODblank]*Set the plate reader-calculated value as the measured OD if the 96-well plate reader performs
automatic calculations.

(2) Calculate the cell viability of a sample using the equation below. Furthermore, calculate thevariability (SD) of tissue replicates.
(3) Record details of steps (1) and (2) above in the MDS 5.

Cell Viability (%)＝

Measured ODsample
Mean Measured ODNC

×100

18

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4. ASSESSMENT
4.1

CONDITIONS FOR A SUCCESSFUL STUDY

The skin irritation test should be considered successful if both of the following criteria have beenmet.
•

Tissue viability: 0.7 ≤ mean OD (A570/650) measured value for negative control ≤ 2.5.

•

Positive control: mean tissue viability for 5% SLS (positive control) ≤ 40%.

•

SD: SD (negative control and positive control) of tissue viability of 3 identically treatedreplicates ≤ 18 %4.2

ASSAY CRITERIA

The criteria for in vitro interpretation are shown below.
The test must be performed 3 times per a sample in total.

Sort the tissue viabilities obtained

from the repeated tests in ascending order, and classify the irritancy based on the median of thosetissue viabilities.
Tissue Viability (primary)Classification

Tissue viability is ≤ 50%

Irritant

Tissue viability is > 50%

Non Irritant[FLOWCHART] ASSESSMENT FLOWCHART(1) Tissue viability in negative control→(either criterion is not met)→

Assay Failure

0.7 ≤ Mean OD measured value ≤ 2.5
Positive control (5% SLS) result should be “irritant”
Mean tissue viability ≤ 40%
SD”
SD (negative control and positive control)of tissue viability of 3 identically treatedreplicates ≤ 18 %↓(All criteria are met)↓
(2) Assessment of test samples (3-time repeated tests: all tests satisfy (1))
The median of the 3 tissue viabilities (%) ≤ 50% →(Yes) →Classified as irritant

↓
(No)
↓
19

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MDS 1:RECEIPT OF LABCYTE EPI-MODEL 24
施設名:

試験名:

試験番号:

Laboratory name

Test name

Test No.

1.

LabCyte EPI-MODEL 24
受領日：
Date received

ロット番号：
Lot No..

使用期限：
Expiration date (MM/DD/YYYY)

付属品：

アッセイ培地 30mL □Accessories

Assay medium, 30mL

（ロット番号：

使用期限：

Lot No.

Expiration date）
(MM/DD/YYYY)

24 ウェルアッセイプレート □24 well assay plate

特記事項 Note2.

アッセイ培地
Assay medium

受領日：
Date received

ロット番号：
Lot No.

使用期限：
Expiration date (MM/DD/YYYY)

特記事項 Note

記録日:

試験担当者:

確認日:

試験責任者:

Date (MM/DD/YYYY)

Operator

Check date (MM/DD/YYYY)

Study director

事務局確認

確認日:

氏名:

Secretariat

Check date (MM/DD/YYYY)

Name

21

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MDS 2:
PRE-INCUBATION OF LABCYTE EPI-MODEL 24 (Section 3.3.1)
施設名:

試験名:

試験番号:

Laboratory name

Test Name

Test No.

1.

アッセイ培地を温め、24 ウェルアッセイプレート第 1 行に 0.5mL ずつ添加する。
Warm up the assay medium and add 0.5mL of the assay medium to the wells of the 1st row on the 24-well assay plate.

アッセイ培地：
Assay medium（ロット番号：

使用期限：

Lot No.

）Expiration date (MM/DD/YYYY)

約 30 分間加温 Warm for 30 min.

□

0.5mL ずつ添加 Add 0.5mL of assay medium to each well
プレート数：□

Number of plates

2.

培養表皮モデルを取り出し、24 ウェルアッセイプレート第 1 行に移す。
Transfer culture inserts to wells in the 1st row on the 24-well assay plate.

LabCyte EPI-MODEL 24：（ロット番号：

使用期限：

Lot No.

Expiration date）
(MM/DD/YYYY)

作業日時：
Time/date executed (MM/DD/YYYY HH:MM)□

培養カップ底面に気泡が無いことを確認
Confirm that there are no bubbles under the cell culture insert.

3.

CO2 インキュベーターに入れ、被験物質暴露まで一晩静置する。
LabCyte EPI-MODEL 24 is cultured in CO2 incubator overnight.

培養開始日時：Time/date of culture start (MM/DD/YYYY HH:MM)

被験物質暴露予定日時：
Planned time/date of exposure to test chemical (MM/DD/YYYY HH:MM)

特記事項 Note

記録日:

試験担当者:

確認日:

試験責任者:

Date (MM/DD/YYYY)

Operator

Check date (MM/DD/YYYY)

Study director

事務局確認

確認日:

氏名:

Secretariat

Check date (MM/DD/YYYY)

Name

22

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MDS 3-1:
APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section 3.1.2, 3.3.2~ 3.3.3)
施設名:

試験名:

試験番号:

Laboratory name

Test name

Test No.

1.

陽性対照物質の調製 Preparation of positive control.
SLS の秤量

mg

Weight of SLS

2.

mL 作業日時：

調製量
Preparation vol.

Operation date (MM/DD/YYYY HH/MM)

アッセイ培地を温め、24 ウェルアッセイプレート第 3 行に 1.0mL ずつ添加する。
Warm up the assay medium and add 1.0mL of the assay medium to the wells of the 3rd row on the 24-well assay plate.

アッセイ培地：（ロット番号：

Assay medium

約 30 分間加温□）Expiration date (MM/DD/YYYY)

1.0mL ずつ添加

Warm for 30 min.

3.

使用期限：

Lot No.

□

作業日時：

Add 1.0mL of assay medium.

Time/date (MM/DD/YYYY HH/MM) executed

被験物質を適用する。
Apply test chemicals to the LabCyte EPI-MODEL 24.

4.

作業開始日時：作業終了日時：Time/date execution started (MM/DD/YYYY HH/MM)

Time/date completed (MM/DD/YYYY HH/MM)

暴露 15 分後、培養表皮モデルを洗浄し、24 ウェルアッセイプレート第 3 行に移す。

After exposure to test chemical for 15 min., wash out the LabCyte EPI-MODEL 24 and transfer the culture inserts to the 3rd row on the 24-well assay plate.

PBS：（ロット番号：

使用期限：

Lot No.

作業開始日時：Time/date execution started）

Expiration date (MM/DD/YYYY)

作業終了日時：(MM/DD/YYYY HH/MM)

Time/date completed (MM/DD/YYYY HH/MM)□

培養カップ底面に気泡が無いことを確認 Confirm that there are no bubbles under the cell culture insert.

5.

被験物質情報 Test chemical information
被験物質コード番号

ロット番号

物理的性状

暴露時間

Lot No.

Physical state

適用量 Test chemical vol.(weight)(秤量記録 measured weight)

適用時刻

Test chemical code No.

Time ofapplication（15 分 15min.）

液体 Liquid

25 L

：

□

液体 Liquid

25 L

：

□

注射用水（陰性対照）
Distilled Water (Negative control)

5%SLS（陽性対照）(Positive control)

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

記録日:

試験担当者:

確認日:

試験責任者:

Date (MM/DD/YYYY)

Operator

Check date (MM/DD/YYYY)

Study director

事務局確認

確認日:

氏名:

Secretariat

Check date (MM/DD/YYYY)

NameExposure time23Version 8.3

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MDS 3-2:
APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION(Section 3.3.2~3.3.3)
施設名:

試験名:

試験番号:

Laboratory name

Test name

Test No.

5.

被験物質情報 Test chemical information
被験物質コード番号

物理的性状

適用量 Test chemical vol.(weight)

適用時刻

暴露時間

Test chemical code No.

Physical state(秤量記録 measured weight)

Time ofapplication（15 分 15min.）

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

液体、粘性液体、固体
Liquid, viscous, solid

6.

Exposure time

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

25µL, (

mg,

mg,

mg)

：

□

CO2 インキュベーターに入れ、42 時間後培養する。
Culture LabCyte EPI-MODEL 24 in CO2 incubator for 42 hrs.

後培養開始日時：
Time/date post-incubation started (MM/DD/YYYY HH:MM)後培養終了予定日時：
Time/date post-incubation completed (MM/DD/YYYY HH:MM)

特記事項 Note

記録日:

試験担当者:

確認日:

試験責任者:

Date (MM/DD/YYYY)

Operator

Check date (MM/DD/YYYY)

Study director

事務局確認

確認日:

氏名:

Secretariat

Check date (MM/DD/YYYY)

Name

24

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MDS 4:MTT ASSAY (Section 3.3.4)
施設名:

試験名:

試験番号:

Laboratory name

Test name

Test No.

1.

2.

MTT 培地の調製 Preparation of MTT medium
調製量mL ロット番号

調製日時：

Preparation vol.

Time/date (MM/DD/YYYY HH/MM) executed

Lot No.

MTT 培地を温め、24 ウェルアッセイプレート第 4 行に 0.5mL ずつ添加する。
Warm up the MTT medium and add 0.5mL of the MTT medium to the wells in the 4th row on the 24-well assay plate.

MTT 培地：（ロット番号：

使用期限：

MTT medium.

Expiration date (MM/DD/YYYY)

Lot No.

約 30 分間加温□Warm for 30 min.

3.

0.5mL ずつ添加

□

）

作業日時：

Add 0.5mL of the MTT medium.

Time/date (MM/DD/YYYY HH/MM) executed

後培養終了後、培養表皮モデルを 24 ウェルアッセイプレート第 4 行に移す。
After post-incubation, the LabCyte EPI-MODEL24 transfer to wells of 4th row of 24-well assay plate.

作業開始日時：作業終了日時：Time/date started (MM/DD/YYYY HH/MM)

Time/date completed (MM/DD/YYYY HH/MM)□

培養カップ底面に気泡が無いことを確認 Confirm that there are no bubbles under the cell culture insert.
4.

CO2 インキュベーターに入れ、3 時間 MTT 反応を行う。
Store LabCyte EPI-MODEL 24 culture overnight in CO2 incubator for 42 hrs.

MTT 反応時間情報 Information on MTT reaction time
被験物質コード番号
Test chemical code No.

ロット
番号
Lot No.

MTT 反応開始時刻

MTT 反応MTT reaction
start time

Time when MTT
reaction ends：

注射用水（陰性対照）

MTT 反応開始時刻

MTT 反応MTT reaction
start time

Time when MTT
reaction ends

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

：

終了時刻

被験物質コード番号
Test chemical code No..

ロット
番号
Lot No.

終了時刻

Distilled Water (Negative control)

5%SLS（陽性対照）(Positive control)

特記事項 Note

記録日:

試験担当者:

確認日:

試験責任者:

Date (MM/DD/YYYY)

Operator

Check date (MM/DD/YYYY)

Study director

事務局確認

確認日:

氏名:

Secretariat

Check date (MM/DD/YYYY)

Name

25

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MDS 5:FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.5)
施設名:

試験名:

試験番号:

Laboratory name

Test name

Test No.

1.

MTT 反応終了後、培養皮膚をピンセットでつまんで取り出し、1.5mL チューブに移す。
After MTT reaction, use forceps to pick up the cultured epidermis from the cell culture insert and put it in a 1.5mL microtube.

□

スカルペルの使用 Did you use a scalpel to cut out the cultured epidermis?
作業日時：
Time/date of execution (MM/DD/YYYY)

2.

イソプロパノール 300µL を入れて培養表皮モデルを完全に浸漬する。
Add isopropanol (300µL) to microtube so that the cultured epidermis is completely immersed in isopropanol.

イソプロパノール

300µL 添加ロット番号
Lot No..

□To add isopropanol (300µL).

□

培養表皮モデルを完全に浸漬 Immersion of the cultured epidermis in isopropanol.
作業日時：
Time/date executed (MM/DD/YYYY HH/MM)

3.

冷暗所で一晩以上静置し、色素を抽出する。For MTT formazan extraction, allow micro tube to stand in a cold and dark space.
冷暗所に静置 Place micro tube in a cold and dark space.
□4.

抽出液（200µL）を 96 ウェルプレートの各ウェルに入れる。
Extract solution (200mL) is transferred to each well on the 96-well plate.

□

抽出液 200µL を 96 ウェルプレートに移す Transfer to the 96-well plate.
作業日時：
Time/date executed (MM/DD/YYYY HH/MM)

96 ウェル割り付け記録 Sample location on 96-well plate.
1

2

A

ブランク
blank

B

注射用水-1Distilled Water-1

C

注射用水-2Distilled Water-2

D

注射用水-3Distilled Water-3

E

5% SLS-1

F

5% SLS-2

G

5% SLS-3

3

4

5

6

7

8

9

10

11

12

H

5.

570nm、及び 650nm の吸光度を測定し、570nm の吸光度から 650nm の吸光度を差し引いた値を測定値とする。Analyze extract OD at 570nm and 650nm, and calculate the OD(570nm-650nm).
570nm、及び 650nm の吸光度を測定 Analyze OD at 570nm and 650nm.
□
測定値（570nm 吸光度-650nm の吸光度）を計算 Calculate the OD(570nm-650nm).
□測定値から生細胞率、及び SD を計算 Calculate cell viability and SD.
□
生細胞率、及び SD を別途配布するファイルに転記 Cell viability and SD are recorded on a separate data sheet.
□
データシートをプリントアウトして裏面に貼付 The data sheet is attached to the back of this sheet.
□
転記ミスがないことを確認 Check for input errors.
□

作業日時：
Time/date executed (MM/DD/YYYY HH/MM)

特記事項 Note

記録日:

試験担当者:

確認日:

試験責任者:

Date (MM/DD/YYYY)

Operator

Check date (MM/DD/YYYY)

Study director

事務局確認

確認日:

氏名:

Secretariat

Check date (MM/DD/YYYY)

Name

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IN VITRO SKIN IRRITATION TEST:HUMAN EPIDERMIS MODEL
Model: LabCyte EPI-MODEL 24

June, 2011

Page 27 of 29

Japan Tissue Engineering Co., Ltd.

REVISION HISTORY
Rev.

Content

Date Revised

Ver.1

1) First version

27/02/2008

Ver.2

1) Revised clerical error.

28/02/2008

Ver.3

1) Revised the post-incubation time and assessment criteria in

17/03/2008

compliance with the EpiSkin method described in “PerformanceStandards for Applying Human Skin Models to in vitro Skin Irritation
Testing”
2) Added photos and figures for instruction.
Ver.41) Added MDS 1~6.

15/05/2008

2) Added instruction and operational steps regarding the IL-1α ELISA
kit.
3) Added subsections “Delivery of LabCyte EPI-MODEL24” and“Instruction For Use of LabCyte EPI-MODEL24” to Section 2.
4) Added the description regarding test chemicals to Section 2.
5) To Section 2, added the description of materials provided by J-TEC
separately from other materials.
6) Stated the specific calculation procedures in Section 3.2.5.2“OPTICAL DENSITY MEASUREMENTS OF EXTRACTS”.
Ver.4.1

1) Moved scalpel from Section 2.4 “MATERIALS PROVIDED BY

21/05/2008

J-TEC” to Section 2.5 “MATERIALS NOT PROVIDED WITH THE
J-TEC KITS”.
2) Removed the description regarding how to execute procedures
alone.
3) Moved IL-1α ELISA reagents from Section 3.1 “PREPARATIONS” to
Section 3.2 “TEST METHOD”.
4) Added a flowchart for the IL-1α ELISA procedures.
5) Changed from “in a cold dark place” to “in a cold dark place (orrefrigerator)” regarding formazan extraction.
6) Added the description of “ultrasonic cleaning equipment or vortex
mixer” as an example of an MTT dissolution method.
7) Changed the exposure time column from entering actual time tocheckboxes on the MDS 3.
Ver.5.0

1) Corrected typing errors in the section number for IL-1α ELISA

27/08/2008

reagents.

27

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IN VITRO SKIN IRRITATION TEST:HUMAN EPIDERMIS MODEL
Model: LabCyte EPI-MODEL 24

June, 2011

Page 28 of 29

Japan Tissue Engineering Co., Ltd.

2) Removed the space for SLS lot numbers on the MDS 3.
3) Removed the space for PBS lot numbers on the MDS 3.
4) Added the space for isopropanol lot numbers on the MDS 5.
5) Added a checkbox about using a scalpel when removing tissues in
the MDS 5.
6) Added the space for IL-1α ELISA kit lot numbers on the MDS 6.
7) Changed the applicable parts of product codes and kit components in
Section 2.2, with the change of IL-1α ELISA kit types to a 96 well test
only.
8) Decreased the volume by half to 10mL and changed the storage
condition from within 1 month to within 24 hours in Section 3.1.2“POSITIVE CONTROL SUBSTANCE”.
9) Added the manufacturers and product codes of the 24-well plate and
96-well plate in Section 2.4 “MATERIALS PROVIDED BY J-TEC”.
10) Added specific time frames for incubation or culturing.
11) Added the conditions for a successful study in Section 4“ASSESSMENT”
12) Changed the specific method of applying liquids in Section 3.2.2.2“APPLICATION OF TEST CHEMICALS”.
13) Added descriptions in English on the MDS Sheets.
14) Changed the application time interval from 1 minute to
1~3minute(s).
15) Numbered figures and flowcharts.
16) Increased the size of spaces for lot numbers on the MDS Sheets.
17) Changed spaces for dates from MM/DD to MM/DD/YYYY.
18) Added director check date, study director, secretariat check dateand name at the end of each MDS.
19) Changed the size of matrixes for sample allocation to a 96-well platein the MDS 5 & 6.
20) Changed the test chemical name to test chemical code in the MDS
3 & 4.
21) Divided the MDS 3 into MDS 3-1 and 3-2, and added spaces for
date, operator, check date, study director at the end of the MDS 3-1,
and spaces for laboratory name, test name and test no. at the
beginning of the MDS 3-2.
Ver. 6.0

1) Removed the descriptions regarding the measurement of IL-1α

27/02/200928

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IN VITRO SKIN IRRITATION TEST:HUMAN EPIDERMIS MODEL
Model: LabCyte EPI-MODEL 24

June, 2011

Page 29 of 29

Japan Tissue Engineering Co., Ltd.

production, since the validation committee decided to use cell
viabilities only as an index for the skin irritancy test at the meeting in2009.
2) Revised the expression “the materials provided by J-TEC” for thevalidation study to that for a standard skin irritancy test preparation.
3) Clearly stated the cell viability equation to use the mean of measured
values.
4) Clearly stated to use the median of cell viabilities from the three-time
repeated tests as assay criteria.
Ver.

1) In order to avoid the possible influence of volatile test chemicals on

6.01

the results of other test chemicals, the types of test chemicals per

23/03/2009

plate was changed from 2 chemicals to just 1 chemical.
Ver.
7.01

1) Test for detecting chemicals that interfere with MTT endpoint was

03/07/2009

added to Section 3.2.

Ver. 7.2

1) Revised clerical error.

30/09/2009

Ver. 8.1

1) Added the description about rational and background as following

30/06/2010

chapters in Section 1.
LabCyte EPI-MODEL 24 SKIN IRRITATION TEST (SIT using
LabCyte EPI-MODEL24)BACKGROUND OF SIT using LabCyte EPI-MODEL24
BASIS OF THE METOD
LIMITATION OF THE METHODBRIEF BASIC PROCEDUREDATA INTERPRETATION PROCEDURE (PREDICTION MODEL)
2) Added photo about chemicals that directly reduce MTT in Section 3.
3) Added the washing protocol more detail in Section 3.
4) Added assessment about SD.
Ver.8.2

1) Changed description about the washing protocol in Section 3.

17/08/2010

2) Changed unit of consumable reagents and vessels from per avalidation study to per a test.
Ver.8.3

1) Changed description more briefly about the washing protocol in

14/06/2011

Section 3.

29