Protocol for in vitro eye irritation using a new 3D
reconstructed human cornea model, MCTT HCETM

CONTENTS

Definitions and Abbreviations ....................................................................................................................................... 1
Protocol Overview ............................................................................................................................................................... 3
Main Protocol........................................................................................................................................................................ 4
1. Background .................................................................................................................................................................. 4
2. Purpose .......................................................................................................................................................................... 4
3. Materials ........................................................................................................................................................................ 4
3.1 RhCE Model: MCTT HCE™ ................................................................................................................................. 4
3.2 Assay quality controls in testing laboratory ............................................................................................. 4
3.3 Test material and vehicle .................................................................................................................................... 5
3.4 Materials and reagent .......................................................................................................................................... 5

4. Methods ......................................................................................................................................................................... 6
4.1 Experimental time schedule .............................................................................................................................. 6
4.2 Preparations of reagents, test chemicals, and instruments ............................................................... 6
4.3 Test for interference of chemicals with WST-1 endpoint and correction procedures .......... 7
4.3.1 Direct staining by test material ................................................................................................................... 7
4.3.2 Reactivity of test substance with WST-1 ................................................................................................. 8
4.3.3 Correction of cell viability............................................................................................................................... 8
4.4 Receipt of MCTT HCETM ...................................................................................................................................... 9
4.5 Pre-incubation ......................................................................................................................................................... 9
4.6 Application and washing of liquid chemicals .........................................................................................10
4.7 Application and washing of solid chemicals ...........................................................................................12
4.8 Post-incubation .....................................................................................................................................................14
4.9 WST-1 Assay...........................................................................................................................................................14

4.10 Optical density measurements of the extracts ....................................................................................15

5. Results........................................................................................................................................................................... 16
5.1 Cell viability.............................................................................................................................................................16
5.2 Criteria of re-test ..................................................................................................................................................17
5.3 Data interpretation ..............................................................................................................................................17

6. Experimental Performance Standards ............................................................................................................ 17
7. List of Data Sheets .................................................................................................................................................. 17MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Definitions and Abbreviations
EIT: Eye Irritation Test
HCE: Human Corneal Epithelium
TEER: Trans Epithelial Electric Resistance
ET-50: Effective Time-50
OD: Optical Density
NC: Negative Control
PC: Positive Control
T: Test substance
ODblank: Diluted WST-1 solution
ODNCraw: Raw OD negative control living tissues
ODPCraw: Raw OD positive control living tissues
ODTraw: Raw OD test substance living tissues
ODNC: OD negative control living tissues (ODNCraw - ODblank)
ODPC: OD positive control living tissues (ODPCraw - ODblank)
ODT: OD test substance living tissues (ODTraw - ODblank)
DPBS: Dulbecco's Phosphate-Buffered Saline without Ca2+ & Mg2+
SDS: Sodium Dodecyl Sulfate
WST-1:

2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
proliferation reagent%NSCliving: Cell viability of living tissue without WST-1 incubation%NSCkilled: Cell viability of killed tissue without WST-1 incubation%NSWST-1killed: Cell viability of killed tissue with WST-1 incubation
mg: Milligram

1

Cell

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

mL: Milliliter
μL: Microliter
mm: Millimeter
nm: Nanometer℃: Degree Celsius
RT: Room temperature%: Percent
hr/hrs: Hour/hours
min: Minute2

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Protocol Overview
MCTT HCE™ models, which passed the quality control (Optical Density (OD) values of negative
control 0.8 - 1.2 ET-50 (the time of exposure to 0.3% Triton X-100 estimated to decrease the viability
by 50%) 17.6 – 41.0 min, and histological examination) of the manufacturer, are delivered to thetesting labs in a 24-well format on agarose gel in refrigerated condition. Upon receipt of the shipment,
culture medium is warmed in a 37°C thermostat for 30 min. As preparation for the pre-incubation
step, 900 μL of the pre-warmed medium is added to each well of a 6-well plate using micropipetteand the HCE model insert is carefully transferred to the wells using forceps. Then the well-plate is
pre-incubated at 37°C, 5% CO2 for 22 ± 2 hr. Following pre-incubation, 40 µL of liquid substanceor solution, or 40 mg of solid substance is topically applied to the upper epithelial surface of themodel insert (0.6 cm2). Then the tissue is incubated again (37°C, 5% CO2 condition) for 10 ± 1 min
or 3 hr ± 5 min depending on the physical state of the test substance (Fig.1). Next, the tissue iswashed to remove the test substance and further incubated for 16 ± 1 hr. Finally, the resulting tissue
viability is evaluated by the WST-1 assay.

[Fig. 1] Overview of the optimized eye irritation test method for MCTT HCETM model3

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Main Protocol
1. Background
This protocol is for the in vitro eye irritation test using reconstructed human corneal epithelium,
MCTT HCETM, as a replacement test for the in vivo Draize eye irritation test using rabbits.

2. Purpose
This protocol describes in vitro eye irritation test using reconstructed human corneal epithelium,
MCTT HCETM, as a replacement test for the in vivo eye irritation test using rabbits. MCTT HCETM
is designed to closely mimic the biochemical and physiological properties of the human cornea. Eyeirritancy of chemicals begins with the penetration of chemicals into the cell layer and is caused bysubsequent damages of the corneal epithelium. Cell viability of the MCTT HCE™ model ismeasured by enzymatic conversion of the vital dye WST-1 [3-(4,5-Dimethyl thiazol-2-yl)-2,5diphenyltetrazolium bromide] into a yellow formazan salt that is quantitatively measured in the
supernatant naturally released from the tissue following incubation with tetrazolium salt. The
purpose of the MCTT HCETM eye irritation test (MCTT HCETM EIT) is to predict the ocular irritancy
of test substances in compliance with the UN GHS classifications for eye irritation.

3. Materials
3.1 RhCE Model: MCTT HCE™

The MCTT HCETM consists of normal human-derived corneal epithelium, which has been
cultured to form a multilayered, highly differentiated model of the human cornea. The MCTT HCETM
consists of organized basal cells, wing cells and squamous cells arranged in patterns analogous tothose found in vivo. The MCTT HCETM tissues are cultured for 14 days on the surface (diameter 12mm, 0.67 cm2) of specially prepared cell culture inserts in Millicell™ (Millipore, USA) culture plates
and shipped in a 24-well format on shipping agarose gel together with the necessary amount of
culture media. In addition, MCTT HCETM tissue can be frozen during shipment. Therefore,
temperature changes during shipment shall be recorded.
The MCTT HCETM system is manufactured according to defined quality control procedures. Allbiological components of the epidermis and the culture medium are tested by the manufacturer toconfirm the absence of viral, bacterial, fungal and mycoplasma contamination. The Effective Time50 (ET-50) value following exposure to Triton X-100 for each MCTT HCETM lot, OD value of thenegative control (PBS) by MTT assay, and histology are provided by the manufacturer within a week
after shipment.
3.2 Assay quality controls in testing laboratory

The absolute OD of negative control (NC) tissues (treated with sterile DPBS) measured by WST1 assay is an indicator of tissue viability of the MCTT HCETM lot delivered to the testing laboratory.
4

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

The assay meets acceptance criterion if the mean OD450 of the NC (DPBS) tissue is ≥ 1.6 and ≤ 3.0.
The reduction in cell viability is the measure for response of the MCTT HCETM tissue to ocular
irritancy. The assay meets another acceptance criterion if the mean viability of PC (SDS 2% or
methyl acetate) is reduced to ≤ 35% of that of the negative control tissue.
3.3 Test material and vehicle

Ingredients must be treated ‘as is’ (not diluted). The most appropriate solvent must be used andthe scientific basis for the selection must be provided. If vehicles other than DPBS are used, theeffects on the cell viability must be considered.
3.4 Materials and reagent

Process

Instruments and reagents
Laminar flow cabinet

Manufacture

Cell incubator
CultureWater bath at 37°C
Extra sterile 6-well plates
BD 353046
Media
From Biosolution
Forceps (Small sterile bluntedged forceps)

Holding insert

DPBS

LONZA 17-512

Negative control, washing test
material

2 (aq) % SDS (Sodium
Dodecyl Sulfate)[CAS: 151-21-3]

Sigma L4509

Positive control

Methyl acetate[CAS: 79-20-9]

Sigma 186325

Positive control
Measuring weight of solidmaterial
Measuring weight of solidmaterial
Treatment of test material
Measuring weight of solidmaterial
Grinding of coarse solidmaterials
All process
Tissue culture & Treatment of
test material
Treatment of test material &washing

Balance
Treatmentof testmaterial

Purpose of use
Sterile condition
Tissue culture at 37°C, 5%
CO2
Warming medium and DPBS
Tissue culture
Tissue culture

Weighing papers
SpatulaMortar and pestle
Auto pipette and tips
Extra sterile 6-well plates
Stop-watches/Timers
Serologicalpipette aid

pipetteandWashing5

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

500 mL beakers
50 mL beakers
Sterile 12-well plates
General laboratory materials(latex gloves, paper towels,
70% EtOH etc.)
WST-1 (3-4,5-dimethyl
thiazole 2-yl 2,5diphenyltetrazolium
bromide)[CAS: 150849-52-8]
WST-1 formazan standard(4-[1-(4-Iodophenyl)-5-(4nitrophenyl)formaz-3-yl]1,3-benzene Disulfonate,
Disodium Salt)[CAS: 150849-53-9]
DPBS
24-well sterile plates

WST-1
assay

96-well plates

Washing
Washing
Washing

BD 353043-

Roche 11 644 807
001
Or 05 015 944 001

Measurement of cell viability

From Biosolution

Evaluation of linearity

LONZA 17-512
BD 353047

Diluting WST-1
Treatment of WST-1
Measuring OD (Optical
Density) value
Separation of formazan
solution from debris
Measuring OD (Optical
Density) value

BD 353072

Centrifuge
ELISA Plate reader (96-well)

4. Methods
4.1 Experimental time schedule

ProtocolDay 1

DeliveryO

Pre-incubation

O

Day 2

Day 3

O

Treatment

O

Washing stepO

Post-incubation

O

O

WST-1 application

O

Reading Optical Density

O

4.2 Preparations of reagents, test chemicals, and instruments

Prepare all reagents just before the commencement of testing. Pre-warmed (37℃) sterile DPBS
should be used as a negative control. Weigh 0.02 g Sodium Dodecyl Sulfate (SDS) and dissolve it in
1 mL sterile DPBS (final concentration; 2%). For a liquid test chemical, aliquot 200 µL (sufficient
6

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

amount for treating 2 different tissues) into an amber vial. For a solid test chemical, aliquot 40 mg
onto weighing paper for each of 2 replicates (in case the chemical might be absorbed into parchment
paper, the chemical should be prepared just before treatment). If solid materials are not finely
powdered, they must be ground into a fine powder using pestle and mortar for weighing purposes.
For materials that are of undefined physicochemical state, such as waxy or crystalline, that deter
weighing, pre-warming in a 37℃ water bath for 15 min may be helpful. After weighing, store thetest substances protected from light. Prepare the diluted WST-1 solution (1:25) in pre-warmed DPBS
(e.g. WST-1 400 µL: DPBS 9.6 mL). The WST-1 solution should be prepared just before treatment
and covered to protect it from light. Also, the linearity of the photometer shall be checked for eachtest with the WST-1 formazan standard provided by Biosolution.

Record on [Data sheet MCTT HCE™_EI/Sheet/007-v1.7, MCTT HCE™_EI/Sheet/008-v1.7][Fig. 2] Measured solid test material in the weighing paper (left); WST-1 solution, after diluting in
DPBS, wrapped with foil to protect it from the light (right).

4.3 Test for interference of chemicals with WST-1 endpoint and correction
procedures

In order to prevent the possible interference with the WST-1 endpoint from colorful chemicals or
chemicals with direct reducing potential, ‘direct staining by test materials’ or ‘reactivity of test
substance with WST-1’ may be checked before the application of test materials. However, when atest substance is determined to be an irritant, the correction is not necessary since corrections always
over-predict the classification by subtracting the cell viability.
4.3.1 Direct staining by test material

1)Preliminary test : Add 1 mL of deionized water and 40 μL (liquid) or 40 mg (solid) of the test
chemical to each eppendorf tube and incubate the mixture in a cell incubator (37°C, 5% CO2,
95% RH) for 60 min.

7

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

2) Take 200 μL of the reacted solution in duplicate and transfer to a 96-well plate. If an OD value
measured at 450 nm is more than 0.1, correction of the cell viability may be necessary (see
4.3.3).
4.3.2 Reactivity of test substance with WST-1

Test substances may reduce WST-1 directly to form formazan. This test may be performed before
the eye irritation test to determine interferences.
1) Preliminary test: Add 40 μl (liquid) or 40 mg (solid) of the test substance to 1 mL of diluted
working WST-1 solution (1:25). Place the mixture in the incubator (37°C, 5% CO2, 95% RH)
for 3 hr. At the end of the exposure time, shake the mixture and evaluate the presence offormazan.
2) If the solution changes color significantly, the test substance is presumed to have thepotential to reduce WST-1. Correction of the cell viability may be necessary (see 4.3.3).
4.3.3 Correction of cell viability for the interference of WST-1 assay

1) When the test substance is only colorant;
The test substance may color-stain the tissue. Conduct the test according to the main protocol
but use DPBS instead of WST-1 solution (NSCliving [Non-specific color in living tissues control]).
This shall be done in duplicate and used as an additional negative control (%NSCliving) for thetest substance in calculating cell viability based on the following equation,

True tissue viability = [%Viabilitytest] - [%NSCliving]2) When the test substance is non-colorant, but WST-1 reducer;
Conduct the test according to the main protocol but use a freeze-killed tissue (NSWST-1 [Nonspecific WST-1 reduction control]) instead of a viable tissue. The freeze-killed tissue has no
metabolic activity, but is capable of absorbing and binding to the test substance. Upon receiptof the tissue, the tissue must be frozen in a 24-well plate at -80℃ for 48 hr. Then transfer thetissue into a 6-well plate containing fresh medium and allow 10 min for it to stabilize. The
resulting cell viability (%NSWST-1) is used to correct cell viability as follows,

True tissue viability = [%Viabilitytest] - [%NSWST-1]

This functional check is not done for every test run, but can be done once, in duplicate, for eachtest substance of concern.

3) When the test substance is both colorant and WST-1 reducer;;
8

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

In this case, the correction needs to be done for both colorant and WST-1 reducer as describedin 4.3.3 1) and 2), but the color interference will be subtracted twice. To achieve this, anotherfunctional check with freeze-killed tissue must be conducted with DPBS instead of WST-1
solution (NSCkilled [Non-specific color in killed tissues control]). Prepare freeze-killed tissue as
described above and conduct the test once, in duplicate, for each test substance. Final cell
viability is calculated as follows,

True tissue viability = [%Viabilitytest] - [%NSWST-1] - [%NSCliving] + [%NSCkilled]

4.4 Receipt of MCTT HCETM

Pre-warm the assay medium to 37℃. Within 5 min of receipt and before beginning incubation,
perform a visual inspection of the inserts to check if tissue surfaces are even, without excess moisture,and there are no air bubbles under the insert. Agarose gel has to be flexible to absorb possible shock
during shipment and must be intact upon receipt of the tissues. Do not use tissues with defects or
fissures, or with excessive moisture on the surface.
Record on [Data sheet MCTT HCE™_EI/Sheet/001-v1.7]

4.5 Pre-incubation

Pipette 0.9 mL of the pre-warmed assay medium into each well of sterile 6-well plates. Remove
the shipped multiwell plate from the refrigerator. Under a sterile laminar flow, open the plastic bag
containing the 24-well plate with epidermal tissues. Carefully take out each insert containing theepidermal tissue. Place the tissue into a 6-well plate prefilled with 0.9 mL medium. Any air bubbles
trapped underneath the insert should be removed. Place the 6-well plates containing the tissues intoa humidified (37℃, 5% CO2) incubator for 22 ± 2 hrs.
Record on [Data sheet MCTT HCE™_EI/Sheet/002-v1.7][Fig. 3] Pre-incubation: Pipette 0.9 mL of the pre-warmed assay medium into each well of a sterile 6well plate (left); then place the tissue into the wells (right)
9

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

4.6 Application and washing of liquid chemicals- Application of the chemical:
Liquid test substances should be applied in a chemical hood since many of them are volatileand odorous. Take 40 μL of the test material with a micropipette and slowly dispense onto
near the center of the cornea model (Fig.4). Rotate the insert to uniformly apply to the wholesurface. When application of the test substance onto all 6-well units is complete, put it in a 5%
CO2 cell incubator at 37°C and incubate for 10 ± 1 min.
Record on [Data sheet MCTT HCE™_EI/Sheet/003-v1.7][Fig. 4] Application of liquids: Pipette 40 μL of liquid test material (left); Dispense directly atop thetissue and spread gently (right).

- Washing: Washing procedure for the 6-wells should be completed within 20 min. DPBS for
washing should be pre-warmed in a 37°C thermostat before use. During washing, place 2-3
cm of gap between the tip of the pipette aid and the tissue and flow DPBS along the well-wall.
Remaining DPBS in-between each washing step is left until the final washing step and shallbe removed all at once.
- Detailed procedures are as below.
1) After 10 ± 1 min of incubation, take the plate from the incubator. Add 4 mL of DPBS, using
pipette aid, into the insert to overflow the material inside, and leave for about 1 min. Then,
flip over the insert using forceps to remove the material inside the insert. The DPBS washinginterval between the wells should be the same as the application interval. For example, if
the substance is applied at 30 sec intervals, washing should also be performed at 30 sec
intervals.
2) Take 10 mL of DPBS using pipette aid to wash the insert. Hold the insert with the forceps
and apply the DPBS to the insert to remove all material inside. The 10 mL of DPBS isapplied over approximately 10 sec (1 mL/ sec). Washing procedure of this test using DPBS
should be maintained at this rate.
3) Place those 6 inserts in each well of a 12-well plate. At 10-sec intervals, add 4 mL of DPBS
to the insert in each well to overflow the material inside, and leave for about 1 min. Then
10

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

grab the insert with forceps and gently shake it 5 times in DPBS to remove debris and place
the inserts on the cover of the 12-well plate.
4) Hold the insert with forceps and rinse inside and outside of the insert thoroughly with 10
mL of DPBS using pipette aid.
5) Finally, remove residual DPBS inside the insert using micropipette and by absorbing withthe sterilized gauze, remove the DPBS on the outside the insert as well.
Record on [Data sheet MCTT HCE™_EI/Sheet/003-v1.7][Fig. 5] 1st washing procedure of liquid material; overflow with sterile DPBS 4 mL staying the tissue
insert for 1 min (left), remove excess of DPBS by gently shaking the insert (right).

[Fig. 6] 2nd washing procedure of liquid material; with sterile DPBS 10 mL, overflowing the tissue
insert.

11

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

[Fig. 7] 3rd washing procedure of liquid material; overflowing DPBS 4 mL in 12-well plate and thenincubate for 1 minute.

[Fig. 8] 4th washing procedure of liquid material; inside and outside of the tissue once with DPBS 10
mL.

[Fig. 9] Blot insert on gauze and remove any rests inside of insert.
4.7 Application and washing of solid chemicals- Application of the chemical:
Start the procedure by taking the cornea model out of the 6-well plate and place it on a coverof the 6-well plate. First of all, wet the surface of the cornea model with 40 μL of DPBS using
a micropipette, then apply 40 mg of the test substance atop and in the center of the cornea
12

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

model. Then, gently shake the insert to allow the substance to spread evenly over the entire
surface (Fig. 10). Once application of test substance is completed in 6 well units, put it in a
5% CO2 cell incubator at 37℃ for 3 hr ± 5 min.
Record on [Data sheet MCTT HCE™_EI/Sheet/003-v1.7][Fig. 10] Application of solids; First, apply 40 μL of DPBS for wetting the surface of the insert (left),
and then apply the weighed test chemical to the insert.

- Washing: The washing process for all 6-wells should be done within 20 min. DPBS for
washing should be pre-warmed in a 37°C thermostat before use. During washing, place 2-3
cm of gap between the tip of the pipette aid and the tissue and flow DPBS along the well-wall.
Leave remaining DPBS in-between each washing step until the final washing, at which point,
remove it all at once.
- Detailed procedures are as follows:
1) After the 3 hr ± 5 min incubation, take the plate out of the incubator. Take 10 mL of DPBS
using a pipette aid. Hold the insert with forceps and apply the DPBS to the insert to remove
materials inside. The DPBS washing interval between the wells should be the same as theapplication interval. If the solid material is not removed during this process, gently wipeaway only the solid material with a sterile swab. Be careful not to scratch or damage thesurface of the cornea model.
2) Repeat step 1).
3) Pick up the insert with the forceps and take 10 mL of DPBS using auto pipette. Turn theinsert back and forth when rinsing with DPBS so that the both sides of the insert are rinsed
thoroughly.
4) To remove the debris, hold the insert with forceps and gently shake the insert more than 5times in a 50 mL beaker containing 30 mL of DPBS. Each time the insert gets rinsed in
DPBS, take the insert out of the beaker and turn upside down to remove the liquid insideinsert. If remaining solid is still noticed by visual inspection, wash it sufficiently at thisstage. If the entire solid is not removed even after washing up to 10 times, do not wash it
any more so as to prevent damaging the cornea. Unwashed beakers should not be used again
for another material.
5) Repeat step 3).
6) Finally, remove residual DPBS inside the insert using a micropipette and by absorbing withsterilized gauze. Remove the DPBS on the outside of the insert as well.
Record on [Data sheet MCTT HCE™_EI/Sheet/003-v1.7]

13

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

[Fig. 11] 1st washing procedure of solid material; rinse inside of the insert with 10 mL sterile DPBS.

[Fig. 12] 4th washing procedure of solid material: completely submerge and shake the insert in 30 mL
DPBS (left); decant DPBS (right).
4.8 Post-incubation

Transfer washed tissue inserts to new 6-well plates pre-filled with 0.9 mL pre-warmed fresh assay
medium. Incubate tissue in the incubator for next 16 ± 1 hr.
Record on [Data sheet MCTT HCE™_EI/Sheet/004-v1.7]

4.9 WST-1 Assay

1) After post-incubation, remove medium inside and outside of inserts using pipette.
2) After removing all medium, transfer inserts into 24-well plates, prefilled with 0.2 mL of WST-1(1:25 diluted). Add 0.1 mL of WST-1 solution inside the insert.
3) Cover the plate with aluminum foil and place in the incubator (37℃, 5% CO2) for 3 hr ± 5 min.
Record on [Data sheet MCTT HCE™_EI/Sheet/005-v1.7]

14

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

[Fig. 13] Remove medium inside and outside of inserts using pipette.

[Fig. 14] Application of WST-1: Fill the 24-well plates with 0.2 mL of WST-1 (1:25 diluted) (left);transfer inserts into each well (center); Add 0.1 mL of WST-1 solution inside inserts (right).

[Fig. 15] Protect the plate from light after application of WST-1 solution.

4.10 Optical density measurements of the extracts1) After WST-1 incubation is completed, collect all reaction solution (inside and outside of theinsert) into the respective well of another plate. Mix the solution to homogeneity by pumping
up and down with the micropipette inside the wells of the 24-well plate. Transfer the resulting
solutions into microtubes and centrifuge at 200 g for 3 min to remove debris.
2) Transfer 200 μL aliquots of the supernatant (duplicate for each tissue) into a 96-well plate
and then check for absence of air bubbles (if air bubbles are present, remove them with a 26
G syringe needle). The recommended plate configuration is given in the spread sheet below.
3) Use working WST-1 solution as blanks. Read OD in a 96-well plate spectrophotometer using
wavelength 450 nm. Print measured OD values for records.
Record on [Data sheet MCTT HCE™_EI/Sheet/006-v1.7]
15

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

4) 96-well plate design for OD measurement is as follows,
1

2

3

4

5

6

7

8

9

10

11

12

A

Blank

Blank

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

B

NC1

PC1

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

Tissue 1

C

NC2

PC2

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

Tissue 2

D

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

E

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

F

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

G

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

H

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

1) Blank: diluted WST-1 solution is used as blank (duplicate)2) NC, Negative Control: Formazan extract solution of DPBS-treated tissues (1 well/tissue, duplicate/NC)
3) PC, Positive Control: Formazan extract solution of PC-treated tissues (1 well/tissue, duplicate/PC)
4) T, Test substance: Formazan extract solution of each test substance-treated tissues (1 well/tissue, duplicate/T)

5. Results
5.1 Cell viability

Cell viability is calculated with the ratio of OD values of treated tissue over that of NC.
1) Blanks (ODblank): Mean value of ODblank for each plate.
2) Negative control (ODNC): Mean OD value of negative control (ODNCraw) minus mean OD
value of blanks (ODblank). Cell viability of negative control should be 100%.
‘ODNC = ODNCraw - ODblank’
3)

Positive control (ODPC): Mean OD value of positive control (ODPCraw) minus mean OD
value of blanks (ODblank).
‘ODPC = ODPCraw - ODblank‘

4) Test substance (ODT): Mean OD value of test substance (ODTraw) minus mean OD value ofblanks (ODblank).
‘ODT = ODTraw - ODblank‘*Cell viability of the test tissue: Calculate the cell viability of a sample using the equation below
Cell Viability (%) =Mean Measured ODTMean Measured ODNC

x 100

* ODNCraw : OD value of negative control from spectrophotometer* ODPCraw : OD value of positive control from spectrophotometer* ODTraw : OD value of test substance from spectrophotometer
16

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

5.2 Criteria for re-test

1) OD value of negative control was below 1.6 or over 3.0.
2) Cell viability of positive control above 35% (CV>35%).
3) Difference of cell viability between duplicate tissues above 20%.
4) Mean value of cell viability was between 30% and 40% for liquid, while 55% and 65% for solid(borderline chemical).

5.3 Data interpretation

Different cut-off values of 35% and 60% indicated the best predictive value for liquid and solid,
respectively. According to the EU and the GHS classification (R/38 Category 2 or No Category), an
irritant is predicted if the mean relative tissue viability of at least two individual tissues exposed tothe test chemical is reduced to 35% for liquid and 60% for solid or below of the mean viability of
the negative controls. The test chemical was defined as a non-irritant if the tissue viability was higher
than 35% and 60% for liquid and solid, respectively. Otherwise, it was determined to be an irritant.

LiquidSolid

Prediction model
Mean tissue viability is ≤ 35%
Mean tissue viability is > 35%
Mean tissue viability is ≤ 60%
Mean tissue viability is > 60%

Classification
Irritant (I) R38
Non-Irritant (NI)
Irritant (I) R38
Non-Irritant (NI)6. Experimental Performance Standards
This Assay should be performed according to Good laboratory practice (GLP).

7. List of Data Sheet-

DeliveryPre-incubation
Treatment
Post-incubation
WST-1 assay
Result of Optical Density
Preparation of reagents
Record of test substances
Record of results for Excel input
17

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Delivery
Test No.

Test Date

20

.

.

8uo

Delivery [ Day 1 – 20.

.

]
Condition of MCTT HCE™ tissues

Receipt of MCTT HCE™ tissues
Date
20

Time
.

.

Recipienthmin# Lot no.:# Number of tissue:

tissue (24 tissue/plate)[Plate A]

Record paper for temperature

1

2

3

4

5

6

2

3

4

5

6

A
B
C
D

[Plate B]
1
A
B
CD# Checking point- air bubbles : ( a )- flat surface : ( f )- extensive moisture on the surface : ( e )- condition of agarose gel : ( c )- no finding: ( - )# Draw a diagonal line on the unused plate diagram

KIT Components
Assay medium
Expiration Date

㎖ x
20

.

=

㎖

.

Study Personnel

Signature

Date

20

.

.

Study Director

Signature

Date

20

.

.

MCTT HCE™_EI/Sheet/001-v1.7

18

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Pre-incubation
Test No.

Test Date

Pre-incubation [ Day 1 ~ Day 2 – 20

.

.

~ 20

20
.

.

.

.

]

Start of pre-incubation time:

Study Personnel(sign)

Date 20.

.

End of pre-incubation time:

Study Personnel(sign)

Date 20.

.

Study Personnel

Signature

Date

20

.

.

Study Director

Signature

Date

20

.

.

MCTT HCE™_EI/Sheet/002-v1.7

19

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Treatment
Test No.

Test Date

Treatment [ Day 2 – 20

.

.

20

.

.

]

Test Chemical Application# No of chemical: total
Code

Treatment time

Liquid(L)/Solid(S)samples with NC and PC
Start of Rinsing

Post-

Remarks

incubation

Well 1

Well 2

Well 1

Well 2

NC

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PC

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Study Personnel

Signature

Date

20

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Study Director

Signature

Date

20

.

.

MCTT HCE™_EI/Sheet/003-v1.7

20

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Post-incubation
Test No.

Test Date
.

~ 20

20
.

.

.

.

Post-incubation [ Day 2 ~ Day 3 – 20.

]

Start of post-incubation time:

Study Personnel(sign)

Date 20.

.

End of post-incubation time:

Study Personnel(sign)

Date 20.

.

Study Personnel

Signature

Date

20

.

.

Study Director

Signature

Date

20

.

.

MCTT HCE™_EI/Sheet/004-v1.7

21

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – WST-1 assay
Test No.

Test Date

WST-1 assay [ Day 3 – 20.

.

Application [# No of chemical: total.

.

]
samples with NC and PC]

End of post-

Start of WST-1

End of WST-1

incubation

incubation

incubation

NC

:

:

:

PC

:

:

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Code

20

OD reading

Remarks:

Study Personnel

Signature

Date

20

.

.

Study Director

Signature

Date

20

.

.

MCTT HCE™_EI/Sheet/005-v1.7

22

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Results of Optical Density
Test No.

Test Date

Results of Optical Density [ Day 3 – 20

.

.

20

.

.

]

[Identification]
1

2

3

4

5

6

7

8

9

10

11

12

A

Blank

Blank

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

B

NC1

PC1

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

Tissue 1

C

NC2

PC2

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

Tissue 2

D

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

E

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

F

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

G

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

H

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

empty

[96-well plate spectrophotometer OD values]

Study Personnel

Signature

Date

20

.

.

Study director

Signature

Date

20

.

.

MCTT HCE™_EI/Sheet/006-v1.7

23

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Preparation of reagents
Test No.
WST-1 [Day 3 - 20

Test date
.

.

20

.

.

]

[WST-1 solution in PBS]
Supplier:

CAS No.:

Lot No.:

Expiration date:

Volume:

PBS volume added:

Study Personnel

Signature

Date

20

.

.

Study director

Signature

Date

20

.

.

MCTT HCE™_EI/Sheet/007-v1.7

24

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Record of test substances
Test substance name or code
Description of Physical consistence (color etc.)
Total weight test substance and vial(g)Storage condition
Receipt date
Date

Storage No
20.

Initial quantity

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

20

.

.

Study director.

Expiration date
Used quantity

Signature

20

Residual quantity

Date

20

.

.

Study personnel.

.

MCTT HCE™_EI/Sheet/008-v1.7

25

MCTT HCETM Eye Irritation Test Protocol (ver. 1.7)

Data Sheet – Record of results for Excel input

26