Version 1.0E, 27 May 2010; Prepared by Kazuhiko Hayashi, Kao Corporation

Short time exposure (STE) test protocol
Index

Description of the test methods
1.

Cells

2.

Preculture3.

Selection of solvents for test substance preparation

4.

Preparation of test substance solutions

5.

Experimental procedures

Data analysis
6.

Calculation of cell viability

7.

Criteria for irritant and non-irritant classifications

8.

Rank classification of eye irritation potential in STE test

Additional information
9.

Culture media and materials

1Test methods
Outline of the STE test procedures is described in Fig. 1.

Fig. 1 STE test methods
Preparation

Experimental procedures

Data analysis

Preculture

Suction removal of culture medium

96-well plates

Addition of test substance solution (200 µL/well)Calculation of cellviability

Still standing for 5 min

Classification as
irritant or non-irritant

3.0 × 104 cells/mL
200 µL/well, 4 days
1.5 × 10 cells/mL
200 µL/well, 5 days
4Suction removal of test substance solution(37°C, 5% CO2)

Wash with PBS (×2)

Solvent Selection• Saline• Saline containing 5% (w/w)
DMSO• Mineral oil

Addition of MTT solution (200 µL/well)

Substance preparation

Removal of MTT solution

Test substancesolutions
5%, 0.5%, 0.05%

Rank classification ofeye irritation potential

Incubation for 2 hr (37°C, 5% CO2)

Addition of 0.04N HCl-isopropanol (200 µL/well)
Still standing for 60 min
Absorbance Measurement (measurement wavelength 570 nm)
MTT: Methyl Thiazolyl Diphenyl-Tetrazolium Bromide

1.

Cells
• Use SIRC cells purchased from ATCC (catalog No. CCL60, lot No. 3981569).

Thecells should be used between 3 weeks and 3 months after the start of cultivation orwithin 25 passages.
• Culture SIRC cells at 37°C under 5% CO2 and humidifying in a culture flask
containing Eagle's MEM supplemented with 10% (v/v) fetal bovine serum, 2 mM
L-glutamine, 50-100 unit/mL penicillin and 50-100 µg/mL streptomycin.

Dissociate

the cells that have become sub-confluent in the culture flask using trypsin-EDTA
solution and subculture in a culture flask or inoculate into 96-well plates.

22. Preculture• Prepare cell suspensions of 3.0 × 104 cells/mL or 1.5 × 104 cells/mL in culture
medium. Preculture the cells for 4 days at 37°C under 5% CO2 and humidifying if
200 µL each of the 3.0 × 104 cells/mL suspension is to be added to each well of the96-well plate; for 5 days if the 1.5 × 104 cells/mL suspension is to be used for thisprocedure.

3.

Selection of solvents for test substance preparation
A flowchart for the procedures from selection of solvents to preparation ofsubstance is shown in Fig. 2.

• First, prepare 5% (w/w) solution of a test substance using saline as solvent andobserve the dissolution pattern of the substance. If the substance dissolves orhomogeneously disperses Note 1), 2), choose saline as the solvent for this substance.
• When the substance does not dissolve or homogeneously disperse in saline, try
saline containing 5% (w/w) DMSO. If the substance dissolves or homogeneously
disperses, choose saline containing 5% DMSO as the solvent for this substance.

3• When the substance does not dissolve or homogeneously disperse in saline
containing 5% (w/w) DMSO, prepare this substance using mineral oil as solvent.
Prepare 5% (w/w) solution of the substance using mineral oil as solvent andobserve the dissolution pattern of the substance. If the substance dissolves orhomogeneously disperses, choose mineral oil as the solvent for this substance. Ifthe substance does not dissolve or homogeneously disperse in mineral oil, do notevaluate this substance in this test.

Note 1): A condition in which a substance is dispersed homogeneously and remains
dispersed for at least 5 minutes
Note 2): Vortex, sonication or proper warm up can increase the solubility.

4.

Preparation of test substance solutions
For assessment of the transferability, test substance is evaluated with 3concentrations of 5%, 0.5% and 0.05%. For the main test in which blinded test
substance is evaluated, 3 concentrations of test substance of 5%, 0.5% and 0.05% areprepared but two concentrations (5% and 0.05%) are tested.
• Prepare sample solutions of the test substances using the solvents selected as
described in Section 3.
• Weigh the test substance in a screw tube and prepare 5% (w/w) solution using theselected solvent.

Then, prepare 0.5% (v/v) and 0.05% (v/v) solutions by serial

10-fold dilutions of the original 5% (w/w) solution.
• Use SDS 0.01% (v/v) saline solution as the positive control.

Prepare SDS 1%(w/w) saline solution, and then prepare SDS 0.1% (v/v) and SDS 0.01% (v/v) saline
solutions by serial 10-fold dilutions starting from the original SDS 1% (w/w)
solution.
• Use the solvents used to prepare sample solutions as the solvent controls.

5.

Experimental procedures• Prior to initiating treatment conditions, familiarize yourself with the treatment
diagram described in Figure 3.
• Preculture the cells to confluency in a 96-well plate.
• Take 0.6 mL (for 0.2 mL × 3 wells) of the prepared test sample using a 1 mL
disposable syringe.

4• Tilt the plate and remove culture medium from each well by suction using a suction
tube (This can be done more easily by connecting a Pasteur pipette to tip of thesuction tube.

Be careful not to touch the well bottom with tip of the Pasteurpipette) .
• Add test samples into wells.

Start stopwatch when the first sample is added andthen add test samples into their designated wells at a rate of 3 wells every 7-10
seconds (see Fig. 3) and record the elapsed time by this.
• Exactly five minutes later, sequentially remove the test samples from all wells bysuction at a rate of 3 wells every 7-10 seconds. Stop stopwatch and record the total
working time by this.
• Carefully add 200 µL each of PBS into the wells from which the test samples havebeen removed, and then remove it by suction. Repeat this manipulation twice towash the inner surfaces of the wells (use of 8-channel pipettor, etc. will facilitate
this wash).
• Thoroughly remove PBS by suction so that not a trace of it will remain in any of thewells.
• Add 200 µL of 0.5 mg/mL MTT (CAS No. 298-93-1) dissolved in culture medium Note3)

to each well and incubate in an incubator (37°C, 5% CO2) for 2 hours.

Note 3): In the case of 21 mg MTT, add 42 mL of MEM culture medium, shake well andlet the mixture stand for 20 minutes.

Shake the mixture well, place it in a 20mL syringe and filtrate through a 0.45 µm microfilter.
Protect MTT from light after weighing until immediately before use.

5Fig. 3 Exposure and Evaluation of Treated Cultures
Evaluation of standard substances
112A
11’’ 2’33’’

SB11’’ 2’33’’

SB11’’ 2’

33’’

S

B

1’

22’’ 3’PC

1’22’’ 3’P

C2’’P

C

1’ 23’

Diagonal lines: PBS to prevent drying (200 µL/well)
1-3’’: Test samples (3 compounds, 3 doses)
P: Positive control (0.01% SDS)
S: Saline
C: Procedural control (culture medium with cells)
B: Blank (culture medium without cells)

H
1.
2.

3.
4.

Remove culture medium.
Add test samples starting at the wells marked 1.
Start stopwatch and then add test sample solutions into designated wells every 7-10 seconds
and record the elapsed time by this.
Five minutes later, suction off the test sample solutions every 7-10 seconds starting at the wells
marked 1. Stop stopwatch and record the total working time by this.
Time course
Wash the wells and add MTT.
5 min
0 min
≤7 min≤2 minWells washed/ MTT added

Removing clture
mediumSuctionTest samples suctioned
every 7-10 seconds starting completed
at the wells marked 1Sampleaddition
completedStopwatch started
Test samples added
every 7-10 seconds
starting at the wells
marked 1

Evaluation of test substances
112A
123PDC123PDC

123PD

C

1’

2’3’S

M

B

1’

2’

3’

S

M

B

1’

2’

3’

S

M

B

H
1.
2.

3.
4.

Diagonal lines: PBS to prevent drying (200µL/well)
1-2’’: Test samples (3 compounds, 2 doses)
P: Positive control (0.01% SDS)
S: Saline
D: Saline containing 5% DMSO
M: Mineral oil
C: Procedural control (culture medium with cells)
B: Blank (culture medium without cells)

Remove culture medium from all wells (except wells for blank).
Add test samples starting at the wells marked 1.
Start stopwatch and then add test sample solutions into designated wells at a rate of 3 wells
every 7-10 seconds and record the elapsed time by this.
Five minutes later, sequentially suction off the test sample solutions at a rate of 3 wells every 7-10
seconds starting at the wells marked 1. Stop stopwatch and record the total working time by this.
Wash the wells and add MTT.

Time course

0 min≤2 min

5 min≤7 minWells washed /MTT added

Removing culture
mediumStopwatch started
Test samples added at
a rate of 3 wells every
7-10 seconds startingat the wells marked 1

Sampleadditioncompleted

Suction completed
Test samples suctioned at a rate of 3wells every 7-10 seconds starting at
the wells marked 1

6• Take out the plate from incubator and discard the liquid in the wells.

Remove theremaining drops completely by lightly tapping the plate onto a paper towel.
• Add 200 µL of 0.04N HCl-isopropanol (isopropanol 96 mL + 1N HCl 4 mL) to eachwell and let the plate stand for 60 minutes.
• Make the extracts in the wells uniform, set the plate onto a plate reader andmeasure absorbance at 570 nm wavelength.

Data analysis
6.

Calculation of cell viability• For each test sample, calculate cell viability (%) as the ratio of the optical density oftest sample to the optical density of solvent control.

Cell viability =optical density of test sample (optical density of test sample - optical density of blank)
× 100
optical density of solvent control (optical density of solvent control - optical density of blank)• In each assay, use 3 wells for each sample concentration and use the average of 3
optical density measurements to calculate cell viability.
• The cell viability of test sample concentration is treated as “zero”, when the averageof 3 well optical densities is less than optical density of solvent control and the cell
viability shows minus.
• Perform 3 independent assays*1 for each sample concentration and use the averageof 3 assays as the final cell viability.

Remark
*1: Each test sample concentration is evaluated with 3 wells on a plate. In case thefollowing 1) to 3)l criteria are satisfied on the plate, the average cell viability of 3
wells is treated as an acceptable assay independently.

Criteria for acceptable test
1) Optical density of the culture medium procedural control (C in Fig. 3) at least 0.3
after subtraction of blank optical density
2) Cell viability for the solvent control at least 80% relative to that for the culture
medium procedural control
3) Cell viability for the positive control (0.01% SDS) within a range of 21.1 to 62.3 %(within a range of mean cell viability 41.7 % ± 10.3 × 2 standard deviations)

74) Standard deviation of the final cell viability derived from 3 independent assays lessthan 15 % for all test sample concentrations*2

Remark*2: In case the standard deviation of cell viability is more than 15 %, additionally other 3
assays are operated and the above criteria 4) is evaluated with independent results
for additional 3 assays.

7.

Criteria for irritant and non-irritant classifications• In the STE test, cell viability is the designated endpoint
to test sample.

after a 5-minute exposure

Substances demonstrating higher than 70% cell viability areclassified as non-irritant (NI) and those demonstrating a cell viability equal to orless than 70% are classified as irritant (I).

8.

Classification

Cell viability

Non-irritant> 70%Irritant≤ 70%

Rank classification of eye irritation potential in STE test
• The procedure for rank classification of eye irritation potential in the STE test is
shown in Fig. 4.
• With the 5% test sample solution, a score of 0 is given when cell viability is higher
than 70% and score of 1 is given when cell viability is not higher than 70%. For
0.05% test sample solution, score of 1 is given when cell viability is higher than70% and a score of 2 is given when cell viability is not higher than 70%. Next, thescores are added up for the 5% and 0.05% test sample solutions and final value iscompared to the rank scale. The substance is classified according the following
scale: Rank 1 = score of 1 = minimally irritant; rank 2 = score of 2 = moderatelyirritant; rank 3 = score of 3 = severely irritant.

8Fig. 4 Rank classification of eye irritation potential in STE
test
STE irritation score
Test sample
solution
5%

Score

Test sample
solution
0.05%

If CV > 70%,

0

If CV > 70%,

1

If CV ≤ 70%,

1

If CV ≤ 70%,

2

Score

CV: Cell viability

5% score + 0.05% score = STE rank

STE rank Eye irritation potential
1

Minimally irritant2

Moderately irritant3

Severely irritant

9Additional Information
9.

Culture media and materials

9-1.

Preparation of culture medium• Add the following materials to 500 mL of E-MEM:-FBS 56 mL-L-glutamine 6 mL-Penicillin-streptomycin 6 mL
9-2.

Precautions in preparing culture medium• If white turbidity is seen in L-glutamine solution, dissolve it in aconstant-temperature bath.
• Prior to the preparation of penicillin-streptomycin solution, lightly shake thecontainer bottle to mix the solution.
• Use disposable pipettes only once throughout the preparation procedure (use two25 mL pipettes for FBS).
9-3.

STE test-related agent and materials
Item

Maker

Item codeNoteATCC

CCL-60

Lot.3981569

Eagle's MEM medium*

Sigma-Aldrich

M2279-500MLFetal bovine serum*

GIBCO

not yet decided

L-glutamine 200 mM*

GIBCO

25030-081

Penicillin-streptomycin*

GIBCO

15140-122

MTT*

Sigma-Aldrich

M-2128

Saline for injection*

OtsukaPharmaceutical081517

Dimethyl sulfoxide(DMSO)*SIGMA-Aldrich

154938Mineral oil*

SIGMA

M5310

PBS tablet

Takara Bio

T900

Sigma-Aldrich

T-3924SIRC cells*

Trypsin-EDTA

10Lot. not yet decided

0.85% NaCl
Penicillin 10,000U
Streptomycin 10mg

Japanese
Pharmacopoeia,20mL, 50 tubes

Mineral oil, Embryo
tested; sterile-filtered

Porcine trypsin 0.5 gEDTA-4 Na 0.2 g in
HBSS 1 L2-propanol

Kanto Chemical

32435-01

1 mol/L hydrochloric acid(1N)

Kanto Chemical

18591-08

Sumilon cell culture flask(middle size)

SumilonMS-21250

Tissue Culture Treated
Polystyrene Sterile

96-well microplate (flatbottom)

Corning3997

Tissue Culture Treated
Polystyrene Sterile

Tissue culture dishCorning

430165

Sumilon serum tube

Sumitomo
BakeliteMS-4601X

Cell scraper

SumilonMS-93170

Terumo syringe

TerumoSS-20ESZ

Microfilter
Millex-HA filter unit

Millipore

SLHA033SS

Falcon tube

BectonDickinson(Falcon)

2070

15 mL tube

Asahi Techno
Glass2314-015

Sumilon pipette

SumilonMS-66050

Disposable pipette

SumilonMS-66250

Eppendorf combi-tipplus(Sterilized)

Eppendorf

30069455

SCC screw tube bottle(white) No. 2As One

7-2110-04

MF-Millipore (cellulose
mixed ester)*Use only product of the same specification. For other reagents and materials,
equivalents regardless of makers thereof are also acceptable.

11