Appendix 3

Protocol for SIRC-CVS cytotoxicity test
Version 3.8
May 24, 2016
Shigenobu Hagino, Ph.D.
Shiseido Research center
2-2-1, Hayabuchi, Tsuzuki-ku, Yokohama-shi, 224-8558, Japan
E-mail shigenobu.hagino@to.shiseido.co.jp

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Contents
1

Purpose ....................................................................................................................................... 3

2

The principle of SIRC cytotoxicity test ......................................................................................... 3

3

Materials ..................................................................................................................................... 3
3.1

Cell line ................................................................................................................................ 3

3.2

Equipment ........................................................................................................................... 3

3.3

Instruments ......................................................................................................................... 4

3.4

Culture medium and reagents ............................................................................................ 4

3.5

Medium ............................................................................................................................... 5

3.6

Crystal violet solution .......................................................................................................... 5

3.7

Test chemicals ..................................................................................................................... 5

3.7.1

Determining solubility or suspensibility of test chemicals in the Medium ................. 5

3.7.2

Preparing test chemicals ............................................................................................. 6

3.7.3

Preparing test chemical dilution series ....................................................................... 7

3.8

4

Reference substances ......................................................................................................... 7

3.8.1

Positive control............................................................................................................ 7

3.8.2

Relative control ........................................................................................................... 7

3.8.3

Negative control .......................................................................................................... 7

Test procedure ............................................................................................................................ 7
4.1

Passaging SIRC cells ............................................................................................................. 7

4.2

Preparing a cell suspension ................................................................................................. 8

4.3

Exposing the cells to a test chemical ................................................................................... 8

4.4

Crystal violet staining .......................................................................................................... 9

4.5

Calculating IC50 .................................................................................................................... 9

4.6

Quality control..................................................................................................................... 9

4.7

Evaluation .......................................................................................................................... 10

5

References ................................................................................................................................. 10

6

List of abbreviations and acronyms ......................................................................................... 11

7

Revision history ......................................................................................................................... 11

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1 Purpose
The Statens Seruminstitut Rabbit Cornea–Crystal Violet Staining (SIRC-CVS) test method has been
designed to be used in a bottom up approach1–3 for distinguishing between ocular non-irritants (NI) andocular irritants (I) by calculating the half maximal inhibitory concentration (IC50) of a chemical substancein Statens Seruminstitut rabbit corneal cells (SIRC) as a measure of cytotoxicity. The results are then used
to predict whether the chemical substance is a non-irritant or an irritant per the UN Globally Harmonized
System of Classification for Labelling of Chemicals (GHS).

2 The principle of SIRC cytotoxicity test
Cytotoxicity is considered a useful index for evaluating the eye irritation potency of chemical substances.
The reason is that corneal epithelium cells are well suited for cytotoxicity tests, because corneal damage
has a significant impact on total eye irritation.4 Cytotoxicity tests are useful for identifying ocular
non-irritants that have almost no effect on the cornea. The Statens Seruminstitut rabbit corneal cell line
used in this test is derived from rabbit corneas. We chose an application time of 72 hours, because in vivo
data from previous research projects5 has shown that, in general, maximal eye irritation caused bychemicals other than acids or alkalis typically occurs within 72 hours of ocular instillation.
In the SIRC cytotoxicity test, crystal violet, which penetrates via a cell membrane treated with methanoland stains biological macromolecules, is used as a means of measuring viable cells. This technique issuitable for many types of cultured cells and produces highly consistent results.5–9 A relative control isalso used to help ensure consistency.10, 11 Not only is the test procedure simple and easy to perform, thetested microplate can be stored and used to verify the test results at any time. In this respect, the SIRCCVS cytotoxicity test is unique among tests used to measure cytotoxicity.
The single greatest disadvantage of this test method is that test chemicals must be dissolved or uniformly
suspended in a liquid medium.

3 Materials
3.1 Cell line
The Statens Seruminstitut rabbit corneal cell line used in this test is derived from rabbit corneas andobtained from the American Type Culture Collection (ATCC No. CCL-60). It is also suitable for storage
frozen in liquid nitrogen. Prior to performing the test, the cells should be checked to ensure the absence of
mycoplasma using a test such as the Venor GeM Mycoplasma Detection Kit (Minerva Biolabs GmbH,
11-1025). The cells are to undergo no more than 35 passages from their purchased stock. (e.g., if the cell
culture starts at passage number 435 and is passaged every four days, it should be disposed of after passage
number 470.) Quality control is to be performed as described in section 4.6.
3.2 Equipment
CO2 incubator, such as the MCO-17AIC from Sanyo Electric Co., Ltd

Clean bench, such as the CCV1300E from Hitachi, Ltd

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

Microplate reader, such as the Benchmark Plus™ from Bio-Rad Laboratories

Inverse phase contrast microscope, such as the Eclipse TS100 from Nikon



Autoclave, such as the BS-325 or SS-320 from Tomy Seiko Co., LtdCentrifuge, such as the 5800 from Kubota CorporationWater bathElectronic chemical balance

Ultrasonic bath sonicatorVortex mixerMagnetic stirrer

Hemocytometer or cell counter, such as the 03-303-5 from Erma Inc.

3.3 Instruments

25-cm2 and 75-cm2 tissue culture flasks, such as the 353108 and 353136 from BD Falcon

96-well flat bottom tissue culture microtiter plates, such as the 353072 from BD Falcon



Storage plates, such as the AB-0765 0.8-mL Storage plate from Thermo Scientific

Multichannel pipettes, micropipettes

Dispenser trays



Tubes



1.5-mL cryotubes



15-mL and 50-mL centrifuge tubes



200-µL, 100-µL, and 5-mL tips for micropipettes

Microplate sealing tapePaper towels, such as the 61000 Kim towel™ from Nippon Paper Crecia Co., LtdWrapping film, such as the Saran Wrap

3.4 Culture medium and reagents

Minimum Essential Medium (MEM)

Fetal Bovine Serum (FBS)
The fetal bovine serum is to be inactivated before use. Inactivate by placing in a water bath at
56°C for 30 minutes. After cooling, store the serum in 56-mL or 28-mL tubes. The serum isstored at −70 or −20°C.

Penicillin/Streptomycin/Amphotericin B (P/S/F) solution(Antibiotic-Antimycotic 100×, GIBCO BRL)

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

Modified PBS, comprising phosphate-buffered saline without calcium or magnesium

0.25% (w/v) Trypsin (1 mmol/L EDTAꞏ4Na)



Dimethyl Sulfoxide (DMSO, CAS Number 67-68-5)
Measured per either weight or volume.

Ethanol (CAS Number 64-17-5)
Measured per either weight or volume.



Crystal Violet (CAS Number 548-62-9)

Methanol (CAS Number 67-56-1)

Sodium Dodecyl Sulfate (SDS, CAS Number 151-21-3)

Triethanolamine (CAS Number 102-71-6)
Purity of 98% or higher.



Hydrochloric Acid (CAS Number 7647-01-0)

Sodium Hydroxide (CAS Number 1310-73-2)

Typical specifications and manufacturers for reagents are shown in Table 1.
3.5 Medium
The medium used in this test (the Medium) comprises MEM supplemented with10% inactivated FBS andabout 1% antibiotic (P/S/F solution). For example, 500 mL of MEM is supplemented with 56 mL of FBS
and 5.6 mL P/S/F. At this time, the concentrations of the antibiotics are 100 U/mL of penicillin, 100 µg/mLof streptomycin, and 250 ng/mL of Amphotericin B.
3.6 Crystal violet solution
A 0.4% crystal violet solution is prepared using methanol.
3.7 Test chemicals
3.7.1

Determining solubility or suspensibility of test chemicals in the Medium

Confirm in advance the solubility or suspensibility of each test chemical in the Medium, using theprocedure shown in Fig. 1. First, determine whether the test chemical can be dissolved or uniformly
suspended in the Medium at a concentration of 10,000 µg/mL (1% w/v). Use a vortex mixer, water bath,or sonicator as necessary. If the test chemical cannot be dissolved or uniformly suspended in the Medium,the next step is to determine whether the test chemical is more easily dissolved in DMSO or ethanol. Next,
dissolve or uniformly suspend the test substance in the more suitable solvent at a concentration of 10,000µg/mL and determine whether that solution can be dissolved or uniformly suspended in the Medium at aconcentration of 10,000 µg/mL. If not, dissolve or uniformly suspend the test substance in the moresuitable solvent at a concentration of 5,000 µg/mL (0.5% w/v) and determine whether that solution can be
dissolved or uniformly suspended in the Medium at a concentration of 10,000 µg/mL. If not, the test

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substance is considered to be outside the applicability domain of the test. These judgments can all be
performed by visually confirming the absence or presence of precipitate in the solution.
1Is the substance soluble or uniformly suspensible
in the medium at a concentration of 10,000 zμg/mL?

Yes

To the test

No
Determine whether the test substance is moresoluble in DMSO or in Ethanol.

Mix the test substance and the more suitable solvent
at a concentration of 10,000 µg/mL.
Is this mixture soluble or uniformly suspensible inthe medium at a concentration of 10,000 µg/mL?

Yes

To the test

No
Mix the test substance and the more suitable solvent
at a concentration of 5,000 µg/mL.
Is this mixture soluble or uniformly suspensible in themedium at a concentration of 10,000 µg/mL?

Yes

To the test

No
The test substance falls outside theapplicability domain for the test.
Figure 1: Determining solubility or suspensibility of test chemicals in the Medium
3.7.2

Preparing test chemicals

After determining an appropriate concentration for each test chemical per the procedure described in
section 3.7.1. When the maximal concentration of a stock test chemical dilution series is 10,000 µg/mL,
once the test chemical dilution series in the microplate is mixed with the Medium containing the SIRC
cells, as described in section 4.3, the final maximal concentration is halved to 5,000 µg/mL (0.5% w/v).
When either DMSO or ethanol is used as a solvent, the final maximal concentration is 5,000 µg/mL (0.5%w/v).
When the maximal concentration of a stock test chemical dilution series is 5,000 µg/mL, the final maximal
concentration in the microplate is 2,500 µg/mL (0.25 w/v%) for the test chemical dilution series and 5,000µg/mL (0.5% w/v) for the solvents. If precipitation is observed in a well at any time after mixing the test
chemical solution and the cells, especially after the 72-hr incubation period, the test data must be rejected.

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3.7.3

Preparing test chemical dilution series

Prepare in duplicate on the microplate an eight-well, two-fold serial dilution for each test chemical, as
shown in Fig. 2: Layout of 96-well microplate.
3.8 Reference substances
3.8.1

Positive control

Use a solution of SDS at a final concentration of 1,000 µg/mL in the Medium as the positive control.
3.8.2

Relative control

Use a solution of triethanolamine at a final concentration of 10,000 µg/mL in the Medium as the relative
control.
3.8.3

Negative control

Use the Medium, a DMSO-medium solution at a final concentration 10,000 µg/mL, or an ethanol-medium
solution at a final concentration of 10,000 µg/mL as the negative control. The negative control shouldmatch the solvent used to dissolve or uniformly suspend the test chemical.

4 Test procedure
4.1 Passaging SIRC cells
Cell culture
1.

Culture SIRC cells in MEM supplemented with 10% FBS and 1% P/S/F (the Medium) at 37°C in
a humidified incubator at 5% CO2 in air. The concentrations of the antibiotics are 100 U/mL of
penicillin, 100 µg/mL of streptomycin, and 250 ng/mL of Amphotericin B.

2.

Remove the Medium from the culture flask, then rinse the SIRC cells twice with 10 mL ofmodified PBS to remove the serum, which is a trypsin inhibiter.

3.

Remove the modified PBS, then add and ensure that all the cells in the culture flask are exposed
to 1.5 to 2.0 mL of 0.25% trypsin solution.

4.

Remove the 0.25% trypsin solution, then incubate the cells as is for two or three minutes at 37°C.
Detach the cells from the inside surface of the flask by tapping. Collect the cells in an appropriate
volume of MEM (10% FBS). Count the cells and prepare a cell suspension at a density of 6 to 8
× 105 cells in 15 to 30 mL of medium. Use this culture to passage the cells.

Freezing and preserving cells
1.

Prepare a mixture of medium and 10% DMSO for freezing and preserving cells. Commerciallyavailable cell preservation solution such as Cellbanker 1or 2 (Juji Field, Inc.) may be used, andthe solution may be either a serum type or non-serum type.

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2.

Add a solution at a density of 1×106 cells/mL to a stock tube and slowly lower the temperature
until frozen. For example, cool the stock tube for 5 minutes in ice, 50 minutes at about −20°C,
and 12 hours at about −70°C before placing it in liquid nitrogen. Commercially available freezing
vessels such as Bicell (Nihon Freezer Co., Ltd) may be used to hold the tube.

3.

The tube containing the cells is then preserved in liquid nitrogen.

Thawing of frozen cells
1.

Immerse the stock tube in hot water at a temperature of 37°C to thaw the frozen cells.

2.

Add 10 mL of the Medium to the cell suspension and centrifuge at 1,000 rpm for 5 minutes.

3.

Remove the supernatant, then add the Medium to prepare the cell suspension.
Passage the preserved cells at least once to confirm appropriate growth.

4.2 Preparing a cell suspension
1.

Remove the Medium from the culture flask, then rinse the SIRC cells twice with 10 mL ofmodified PBS to remove the serum, which is a trypsin inhibiter.

2.

Remove the modified PBS, then add and ensure that all the cells in the culture flask are exposed
to 1.5 to 2.0 mL of 0.25% trypsin solution.

3.

Remove the 0.25% trypsin solution, then incubate the cells as is for two or three minutes at 37°C.

4.

Detach the cells from the inside surface of the flask by tapping.

5.

Collect the cells in an appropriate volume of MEM (10% FBS) with a pipette.

6.

Count the cells and prepare a cell suspension at a density of 2 × 105 cells/mL.

4.3 Exposing the cells to a test chemical
1.

Prepare 100 µL of modified PBS and the negative control as well as 100 µL of the serial dilutionsof the test chemical, positive control, and relative control in a 96 well microplate, as shown in Fig.
1.

2.

Add 100 µL of the 2 × 105 cells/mL cell suspension to the wells, as shown in Fig. 2.

3.

Seal the microplate to prevent contamination from volatile test chemicals. Wrapping film may be
used for this purpose. The six measurements described in steps (1)–(6) of section 4.6 Quality
Control are to be used to verify that there is no contamination of other wells by volatile test
chemicals. The criterion for toxic effect is the same as that for quality control. If contamination isfound, the test is to be redone at a lower concentration.

4.

After mixing the test chemical and the cell suspension, allow to stand for 20 minutes on a clean
bench. Once the cells adhere to the bottom of the wells, the microplate is moved to the incubator.

5.

Incubate for about 72 hours at 37°C and 5% CO2 in air.

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4.4 Crystal violet staining
1.

After incubation, remove the Medium containing the test chemicals by gently but quickly turning
the microplate upside down.

2.

Add 200 µL of modified PBS and shake gently to rinse the cells, then remove the modified PBS
by gently but quickly turning the microplate upside down. Repeat this procedure twice.

3.

Add 100 µL of crystal violet methanol solution to each well and allow to stand for 30 minutes.

4.

After the staining, remove the crystal violet methanol solution by gently but quickly turning the
microplate upside down. Wash the cells thoroughly with tap water and blotted away any residual
water with a paper towel.

5.

After drying, measure the optical absorbance at 588 nm with an automatic microplate reader. Any
nearby wavelength for which equivalency can be demonstrated is suitable for measurements.

4.5 Calculating IC50
Absorbance in the negative control wells, which contain no test chemical, minus the absorbance of theblank is considered to be 100%, and the percentage of absorbance for the mean of two wells is calculatedon this basis. Cell viability is a percentage calculated by dividing the mean absorbance of two wells at thesame concentration minus the absorbance of a blank well by the mean absorbance of all negative control
wells minus the absorbance of a blank well.
IC50 is the concentration at which the growth of cells was inhibited to 50% of the control and calculated
as follows using two concentrations around the predicted concentration of 50% cell viability.
Log IC50 = [(50 − y1)log x2 − (50 − y2)log x1]/(y2 − y1),where x1 is low concentration, x2 is high concentration, y1 is cell viability at low concentration, y2 is cell
viability at high concentration, and log means the common logarithm.
If cell viability is greater than 50% at maximal concentration of 5,000 µg/mL, the result for that test
chemical is IC50 > 5,000 µg/mL. Also, if the cell viability is less than 50% at a minimal concentration of
39.1 µg/mL, the result for that test chemical is IC50 < 39.1 µg/mL. IC50 at other maximal and minimal
concentrations of test chemicals are expressed in the same manner.
If multiple concentrations of a test chemical yield a 50% cell viability, use the lowest value of IC50.
In the Excel spreadsheet, cell viability is rounded to the nearest tenth.
4.6 Quality control
Quality control of the SIRC cytotoxicity test is performed by taking six measurements, which must satisfy
the following criteria. Failure to satisfy the criteria means that the test substance must be retested. In
particular, if a volatile test chemical fails to satisfy the criteria, it must be retested at a lower concentration.
1.

The absolute OD obtained from the negative control is an index of the normal proliferation of
SIRC cells seeded at a concentration of 2 × 104 cells/well and incubated for 72 hours. The mean

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OD of the negative control (right and left wells) must be greater than 0.4 for the test data to beconsidered valid.
2.

Sodium dodecyl sulfate (SDS) is used as a positive control. The IC50 of SDS should be between
77.7 and 258.7 µg/mL when tested using the standard protocol. This criterion must be satisfied
for the test data to be considered valid.

3.

Triethanolamine is used as a relative control (See Annex 1.). The IC50 of triethanolamine should
be between 1,000 and 2,500 µg/mL when tested using the standard protocol (See Annex 2 ). This
criterion must be satisfied for the test data to be considered valid.

4.

Any discrepancy between the two dilution series of the test chemical is to be reviewed. The IC50
of both the first series and the second series must be within 20% of the mean IC50 of the twodilution series together. This criterion must be satisfied for the test data to be considered valid.
The minimum value for IC50 is 39.1 µg/mL and the maximum value is 5000 µg/mL. IC50 at othermaximal and minimal concentrations of test chemicals are expressed in the same manner. These
values of IC50 are only used for quality control calculations.

5.

The difference between left and right wells of the negative control should be reviewed to confirm
systematic quality. The mean OD of the left side and the mean OD of the right side should bewithin 15% of the mean OD of both sides combined. This criterion must be satisfied for the test
data to be considered valid.

6.

The two test results adopted for making a prediction must be checked for equality. The higher of
the two IC50 values of the two positive controls (SDS) must be no more than twice as large as thelower of the two values. (The higher value ÷ the lower value ≦ 2)

4.7 Evaluation

Eye irritation potency of the test chemical is predicted using triethanolamine as a relative control (SeeAnnex 1.). Triethanolamine is classified No Category under GHS, and using this as a reference, a test
chemical is identified as negative (No Category) when the IC50 is higher than or equal to that oftriethanolamine and is identified as positive (Category 1 or 2) when the IC50 is lower than that oftriethanolamine. The test is performed twice. If the results of the two tests are different, a third test is
performed and the data of the two tests with the same result are adopted for evaluating. If discrepancies
between three results must be reviewed, the test is repeated three times.

5 References
1.

Scott, L. et al., Toxicol. In Vitro, 24(1), 1-9 (2010).

2.

Hagino, S. et al., ATLA, 36, 641-652 (2008)

3.

Hagino, S. et al., ATLA, 38, 139-152 (2010)

4.

Itagaki, H. et al., Toxicol. in Vitro, 5, 139-143 (1991).

5.

Ohno, Y. et al., Toxicol. in Vitro, 13, 73 (1999).

6.

Saotome, K. et al., Toxicol. in Vitro, 3, 317-321 (1989).

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250

7.

Itagaki, H., AATEX, 3, 182-190 (1995).

8.

Ohno, Y et al., AATEX, 3, 123 (1995).

9.

Tani, N., Toxicol. in Vitro, 13,175 (1999).

10.

Guidance for evaluation of eye irritation of cosmetic ingredients using alternative method (Draft
document by the study team supported by Ministry of Health and Welfare), AATEX,5,Suppl.,
Guideline Draft1-3 (1998).

11.

Ohno, Y., ATLA, 32, Supplement 1, 643-655, 2004.

6

List of abbreviations and acronyms°C

degrees Centigrade

ATCC

American Type Culture Collection

DMSO

Dimethyl Sulfoxide

EPA

United States Environmental Protection Agency

FBS

Fetal Bovine Serum

GHS

Globally Harmonized System of Classification and Labelling of Chemicals

IC50

half maximal inhibitory concentrationI
JaCVAM
MEM

Irritant
Japanese Center for the Validation of Alternative Methods
Minimum Essential Medium

NI

Non Irritant

OD

Optical density

Modified PBS
SDS

Phosphate-Buffered Saline without calcium or magnesium
Sodium Dodecyl Sulfate

SIRC cells

Statens Seruminstitut Rabbit Corneal cells

SIRC-CVSStatens Seruminstitut Rabbit Cornea–Crystal Violet Staining

7 Revision history(1) The revision of ver.1 – ver.1.71 that is the same as ver.1.71j and ver.1.71e, is shown by the green
character in ver. 2.13. The protocol of the ver.1 was used for evaluating 68 chemicals at Shiseido ResearchCenter in 2009-2010 and was subjected to the peer review by JaCVAM.
(2) The revision after ver.1.71 is shown by the blue character in ver. 2.13.
(3) The revision from ver.2.07 to ver.2.08:

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In 4.7.(4), “The difference between two dilution series of the substance should be confirmed. The IC50sof the first series and the second series should be within + 20% of the mean IC50 of two series （the mean
+ 20%）, respectively.” was changed to “The difference between two dilution series of the substanceshould be confirmed. The IC50s of the first series and the second series should be within + 20% of themean IC50 of two series(the mean of the first IC50 and the second IC50), respectively.”(4) The revision form ver.2.08 to ver.2.09:
In 4.3.(3), “The five measurements (4.7.(1)-(5)) of the quality control should be used for checking wether
the volatile substance has an effect on other wells. The criterion of the toxic effect is the same as that of
the quality control. When the volatile substance has an effect on other wells, the retest should be performed
using dilution.”(5) The revision from ver.2.09 to ver.2.11:“SIRC cytotoxicity test” was changed to “SIRC-CVS cytotoxicity test”. “Table 1” of 3.7.1. was changed
to “Figure 1”.
(6) The revision from ver.2.11 to ver.2.12:
The version was added to title. SIRC-CVS was added to list of abbriviations and acronyms of 6. “Figure
1. Layout of 96 well microplate“ was changed to “Figure 2. Layout of 96 well microplate”. “Figure 2.
Addition of cell suspension” was changed to “Figure 3. Addition of cell suspension”(7)The version from ver.2.12 to ver.2.13:
In 3.7.2., “Futhermore, the data of the well with precipitation or so on at anytime after the mix of thesubstance and the cell should be reject for unsuitable suspension” was changed to “Furthermore, the data of the well with precipitation or so on at anytime after the mix of the substance andthe cell, especially after 72 hr incubation, should be rejected for unsuitable suspension”.
The address of the author, “2-12-1, Fukuura, Kanazawa-ku, Yokohama-shi, 236-8643 Japan” was changed
to “2-2-1, Hayabuchi, Tsuzuki-ku, Yokohama-shi, 224-8558, Japan”.
(8)The revision from ver.2.12 from ver.3.1:
The version and the date were renewed.
In 1., “and United States Environmental Protection Agency (EPA)” was deleted.
In 3.1, “（e.g. When the cell culture starts at passage number of 435 and is maintained by two passages
per one week, it should be used within passage number of 458）” was added.
In 3.4., ”The manufacturer and so on of the reagent is shown in the Table 1.” was changed to “The exampleof the manufacturer and so on of the reagent is shown in the Table 1.”.
In 3.7.1., “The solubility of the substance in the medium should be confirmed in advance.” was changed
to “The solubility of each substance in the medium should be confirmed in advance, using the procedureshown in Fig. 1.”.
In figure 1, “Select the solvent on the basis of the examination of the solubility in DMSO or Ethanol.” waschanged to “Examine which solvent is more soluble, DMSO or Ethanol，and select appropriate solvent.

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In 4.6., “For EPA standard, the test substance is judged as negative (Category 4) when the IC50 is higher
than that of triethanolamine, and is judged as positive (Category 1-3) when the IC50 is lower than or equal
to that of triethanolamine （see table 3 and 4 of annex 1）.” was deleted.
In table 1., “The manufacturer and so on of the reagent” was changed to “The example of the manufacturer
and so on of the reagent” . Also, lot numbers were deleted from remarks.
In annex 1, “The same examination that the in vivo evaluation is performed by EPA classification is shownin table 2. The in vivo results are discriminated between category 4 and others (category 1-3) of the EPA
standard. Triethanolamine is classified as category 3. Therefore, when the IC50 of future test substanceis higher than that of triethanolamine, it should be evaluated as a non irritant. If the IC50 of the test
substance is lower than or equal to that of triethanolamine, it should be evaluated as an irritant.” wasdeleted.
Table 2 and Table 4 of annex 1 were deleted and table number was moved up.
(8)The revision from ver.3.1 to ver.3.2:
In 4.7. (4), “Treatment of IC50 expressed with inequality sign is performed in the same manner.” wasadded.
(9)The revision from ver.3.2 to ver.3.3:
In “1.Purpose”, “as a bottom up approach（Scott et al., 2010）” was added.
In “2. The principle of SIRC cytotoxicity test”，”The relative control was additionally used to obtain theinvariable results (Ohno, 2004).“ was added.
In triethanolamine of “3.4. Culture medium and reagent”, “It should be used that of Purity>=98.0%.” wasadded.
In 3.7.2., overlap with 3.7.1 was removed. “The test substance is solved or uniformly suspended withmedium at a concentration of 10,000 µg/mL (1% w/v). It is solved or uniformly suspended using vortex
mixer, waterbath and sonicator when it finds necessary. DMSO or ethanol is used for solving or
suspending if needed. The concentration of DMSO or ethanol is 10,000µg/mL (1% w/v) in the initialsubstance solution. The solvent selection is medium, DMSO in medium and ethanol in medium in order.
In addition, the concentration of the substance is decreased to 5,000 µg/mL (0.5% w/v) for suspending
when it finds necessary. The substance that is not suspended homogeneously is judged as an inapplicable
substance for this test.”was deleted. And, “The test substance is solved or uniformly suspended at
appropriate concentration by the procedure described in 3.7.1.” was added.
In “4.6.Evaluation”, “on the basis of the in vivo data by Ohno et al. “ was deleted.
In “5.References” and the text, the reference numbers were added. The format of reference was changed.
The references of the following paper were added.
Ohno, Y., ATLA, 32, Supplement 1, 643-655, 2004.
Scott, L. et al., Toxicol. In Vitro, 24(1), 1-9 (2010).
The reference of the following paper was deleted.

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Ohno, Y. et al., In Vitro Toxicol.,7, 89 (1994)
Table 2 of annex 1 and the related sentences were deleted.
In reference of annex 1, the format of references was changed.
(10)The revision from ver.3.3 to ver.3.4:
In 2., “The application time of 72hr is set with consideration of time (within 72hr) from ocular instillation
to maximal eye irritation for general chemicals except acid or alkali, on the basis of in vivo data at theprevious research project5).” was added.
In 2., “The SIRC cytotoxicity test procedure is based on the measurement of viable cells stained by crystal
violet.” was changed to “The SIRC cytotoxicity test is based on the measurement of viable cells stainedby crystal violet, which penetrates via cell membrane and stains biological macromolecules.”In 2., “On the other hand, the disadvantage of this method is to be confined to test substances which aresolved or uniformly suspended in the medium.” was added.
In 4.5., “If the multiple concentrations showing 50% cell viability were obtained from one substance, thelowest IC50 should be adopted.” was added.
(11)The revision from ver.3.4 to ver.3.5:
In 2., “The SIRC cytotoxicity test is based on the measurement of viable cells stained by crystal violet,
which penetrates via cell membrane and stains biological macromolecules” was changed to “The SIRC
cytotoxicity test is based on the measurement of viable cells stained by crystal violet, which penetrates via
cell membrane treated with methanol and stains biological macromolecules.”.
In 3.1., “The cells should be used during 3 months after the start of cultivation” was changed to “The cells
should be used within 35 passages from their purchased stock”.
In 3.4., the explanation of Phosphate-Buffered Saline（-）,“Calcium and magnesium are removed from
PBS” was added.
4.4 was reinstated from mistaken deletion in ver.3.4.
In (1) of 4.7., “seeded at the concentration of 1x104 cells/well” was changed to “seeded at theconcentration of 2x104 cells/well”.
(12)The revision from ver 3.5 to ver.3.6:
In 4.5., “Treatment of IC50 expressed with inequality sign is performed in the same manner.” was added.
The order of 4.6.Evaluation and 4.7. Quality control was changed to that of 4.6. Quality control and 4.7.
Evaluation.
In (4) of 4.6., “The treatment to IC50 expressed with equality sign are only used at the calculation for
quality control.” was added.
(13) The revision from ver.3.6 to ver.3.7:
In 4.7, “ of GHS standard” was revised to “of UN GHS classification system”.

Page 14 of 21254

In 3.7.1., “and has no test.” was revised to “and is not judged as testable.”(14) The revision form ver.3.7 to ver 3.8
In 3.8, many minor revisions for readability were made by a native-English speaker. (see Word file)

Page 15 of 21

255

Table 1. Typical reagents and their manufacturers
Reagent or Medium

Manufacturer

Catalog number

Notes

MEM (Minimum Essential Medium)
GIBCO
Code No. 11095
Fetal Bovine SerumGIBCO
REF No. 26140-079
Penicillin-Streptomycin-Amphotericin (100x)
GIBCO/BRLREF No. 15240-062
Phosphate-Buffered Saline (modified PBS)
Nissui
Code No. 05913
Wako
Cat No. 209-16941
0.25% (w/v) Trypsin (1mmol/L EDTA･4Na)
Dimethyl sulfoxide (DMSO)
Kanto
Cat No. 2950-1B
Ethanol
Wako
Cat No. 057-00456
Crystal violet
Wako
Cat No. 031-04852
Methanol
Wako
Cat No. 131-01826
Sodium Dodecyl Sulfate
Wako
Cat No. 191-07145
Triethanolamine
Kanto
Cat No. 40268-00
Products of the same specification from the specified manufacturer may be used for Minimum Essential
Medium, Fetal Bovine Serum, Penicillin-Streptomycin-Amphotericin, Sodium Dodecyl Sulfate, andTriethanolamine. Equivalent products from other manufacturers are acceptable for other reagents.
Nissui: Nissui Pharmaceutical Co., Ltd
Wako: Wako Pure Chemical Industries, Ltd.
Kanto: Kanto Chemical, Co., Inc.

Page 16 of 21

256

Figure 2: Layout of the 96-well microplate
1

2

3

4

5

6

7

8

9

10

11

12

A

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

B

PBS

NC

S1

S2

S3

S4

S5

S6

S7

S8

NC

PBS

C

PBS

NC

S1

S2

S3

S4

S5

S6

S7

S8

NC

PBS

D

PBS

NC

R1

R2

R3

R4

R5

R6

R7

R8

NC

PBS

E

PBS

NC

R1

R2

R3

R4

R5

R6

R7

R8

NC

PBS

F

PBS

NC

P1

P2

P3

P4

P5

P6

P7

P8

NC

PBS

G

PBS

NC

P1

P2

P3

P4

P5

P6

P7

P8

NC

PBS

H

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS

PBS: 200 µL of modified PBS
NC: The Medium, a DMSO-Medium solution at a concentration of 10,000 µg/mL, or an ethanol-Medium
solution at a concentration of 10,000 µg/mL
S: Eight-well, two-fold serial dilution of the test chemical (100 µL per well)
R: Eight-well, two-fold serial dilution of the relative control (100 µL per well)
P: Eight-well, two-fold serial dilution of the positive control (100 µL per well).
Serial dilution of the test chemical is made using the Medium, a 10,000-µg/mL concentration of DMSOMedium solution, or a 10,000-µg/mL concentration of ethanol-Medium solution. Serial dilution of thepositive and relative controls are made using the Medium.
Figure 3. Addition of cell suspension
1

2

3

4

5

6

7

8

9

10

11

12

A
B
C
D
E
F
G
H

■ Cell suspension (100 µL)
Page 17 of 21257

Annex 1 Rational for using triethanolamine as a reference control
Triethanolamine was selected as a relative control substance of the SIRC cytotoxicity test for
distinguishing between ocular non-irritants (NI) and irritants (I) per the Globally Harmonized System ofClassification and Labeling of Chemicals (GHS). It is one of the substances used in the previous validation
study that was performed by a Research Grant for Health Sciences, MHW and Japanese Cosmetic Industry
Association, and reported by Ohno et al.1 and Tani et al.2 Also, it is readily available commercially, issoluble in the Medium, and has been studied extensively, with plenty of useful cytotoxicity and in vivo
data available for evaluating eye irritation potency.
In selecting the relative control substance, accuracy and other characteristics for distinguishing between
NI and I were checked for every available substance from previous validations. As a result of thiscomparative study, triethanolamine was selected as a relative control substance because of a relatively low
instance of false negatives, a high accuracy except for substances which for which cytotoxicity could not
be quantified (10000 < etc.), and clarity of categorization (for example, alcohol) for substances yielding
false negative, as shown in table 1. Identification of substances likely to result in false negatives isimportant from the perspective of minimizing the risk of eye irritation.
References
1) Ohno, Y. et al., Toxicology in Vitro 13, 73-98 (1999)
2) Tani, N. et al., Toxicology in Vitro 13, 175-187(1999)

Page 18 of 21

258

19

259

14800 <
11600
10000 <
10000 <
9330 <
8880 <
2090
1230
1190
963
952
868
721
699
474
392
297

NI
NI
NI
1 or 2ANI
1 or 2ANI
1
1 or 2ANI
1 or 2A
1 or 2A
1 or 2A
1, 2A, or2B
NI
1
1 or 2A

Silicic anhydride

Glycerin

Isotonic sodium chloride solution

Triethanolamine

Lactic acid

Sodium salicylate

Glycolic acid*Acetic acid*

2-Ethylhexyl p-dimethylamino benzoate

Calcium thioglycolate

Acid red 92

Diisopropanolamine*

Polyoxyethylene sorbitan monooleate (20E.O.)

Benzyl alcohol

Butanol*

Isopropyl myristate

Ethanol

35300 <NI

Polyethylene glycol 400

297

392

474

699

721

868

952

963

1190

1230

2090

8880

9330

10000

10000

11600

14800

35300

8

8

8

7

7

7

7

7

6

6

6

5

5

4

4

3

2

1

10

9

8

8

7

6

5

4

4

3

2

2

1

1

1

0

0

0

15

16

17

17

18

19

20

21

21

22

23

23

24

24

24

25

26

27

1

1

1

2

2

2

2

2

3

3

3

4

4

5

5

6

6

6

68

71

74

71

74

76

79

82

79

82

85

82

85

82

82

82

82

82

Table 1 The correlative evaluation on the basis of the previous validation study data for selecting relative control substance- GHS classification Key: TN: true negative, FN: false negative, TP: true positive, FP: false positive
Ranking
TN
FN
TP
FP
Acc
Substances
GHS classification
SIRC(%)
based on In vivo
cytotoxicity: IC50
NB2
testingNB1(µg/mL)

20

260
218
207
181
168
140
120
(Data from 4 labs)
67.6
38.4
37.8
19.0
12.1
9.62
2.59
(Data from 4 labs)
1.67
1.58

1 or 2ANI
1 or 2A
1 or 2A
1 or 2A
1 or 2A
1 or 2A
1 or 2A
1
1 or 2A
1 or 2A
1 or 2A
1 or 2A
1
1

m-Phenylenediamine

Di (2-ethylhexyl) sodium sulfosuccinate*

Sodium lauryl sulfate*

Sodium hydrogenated tallow L-glutamate*

Potassium laurate*

Chlorhexidine gluconate (20% solution)*

Polyoxyethylene octylphenylether (10 E.O.)*

Benzalkonium chloride*

Domiphen bromide*

Monoethanolamine*

Cetyltrimethylammonium bromide*

Cetylpyridinium chloride*

Stearyltrimethylammonium chloride*

Page 20 of 21

The in vivo results for “as is” applications were predicted from data taken with 10% concentrations.

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

*

0

1

2

3

4

5

6

7

8

9

10

11

12

13

13

14

Data for the SIRC cytotoxicity test are the mean IC50 (ug/mL) from five or more laboratories, except for one part.

25

24

23

22

21

20

19

18

17

16

15

14

13

12

12

11

NB2

9

9

9

9

9

9

9

9

9

9

9

9

9

9

8

8

The Draize eye test results do not always discriminate between GHS Category 1 and Category 2 when data was recorded on day 21.
Data was recorded on day 14.

1.58

1.67

2.59

9.62

12.1

19

37.8

38.4

67.6

120

140

168

181

207

218

286

NB1

Distearyldimethylammonium chloride

Methyl p-hydroxybenzoate

286

1 or 2A

Sucrose fatty acid ester

26

29

32

35

38

41

44

47

50

53

56

59

62

65

62

65

21

261

Page 21 of 21

The IC50 range of triethanolamine, 1,000–2,500 µg/mL, is based on the mean +2 standard deviations from the data (n=144).

Annex 2 The basis for the set IC50 range of triethanolamine as a reference control