Section 4

Health effects

Test Guideline No. 491

Short Time Exposure In Vitro Test
Method for Identifying i) Chemicals
Inducing Serious Eye Damage
and ii) Chemicals Not Requiring
Classification for Eye Irritation or
Serious Eye Damage

26 June 2020

OECD Guidelines for the

Testing of Chemicals
 OECD/OCDE 491
Adopted:
26 June 2020

OECD GUIDELINE FOR TESTING OF CHEMICALS

DRAFT UPDATED TEST GUIDELINE 491: SHORT TIME EXPOSURE IN

VITRO TEST METHOD FOR EYE HAZARD POTENTIAL

INTRODUCTION

1. The Short Time Exposure (STE) test method is an in vitro method that can be used
under certain circumstances and with specific limitations for hazard classification and
labeling of chemicals (substances and mixtures) that induce serious eye damage as well as
those that do not require classification for either serious eye damage or eye irritation, as
defined by the United Nations (UN) Globally Harmonized System of Classification and
Labeling of Chemicals (GHS) (1).
2. For many years, the eye hazard potential of chemicals has been evaluated primarily
using an in vivo rabbit eye test (TG 405). It is generally accepted that, in the foreseeable
future, no single in vitro alternative test will be able to fully replace the in vivo rabbit eye
test to predict across the full range of serious eye damage/eye irritation responses for
different chemical classes. However, strategic combinations of alternative test methods
used in a (tiered) testing strategy may well be able to fully replace the rabbit eye test (2).
The top-down approach is designed for the testing of chemicals that can be expected, based
on existing information, to have a high irritancy potential or induce serious eye damage.
Conversely, the bottom-up approach is designed for the testing of chemicals that can be
expected, based on existing information, not to cause sufficient eye irritation to require a
classification. While the STE test method is not considered to be a complete replacement
for the in vivo rabbit eye test, it is suitable for use as part of a tiered testing strategy for
regulatory classification and labeling, such as the top-down/bottom-up approach, to
identify without further testing (i) chemicals inducing serious eye damage (UN GHS
Category 1) and (ii) chemicals (excluding all solid chemicals other than surfactants) that
do not require classification for eye irritation or serious eye damage (UN GHS No
Category) (1) (2). However, a chemical that is neither predicted to cause serious eye
damage (UN GHS Category 1) nor UN GHS No Category (does not induce either serious
eye damage or eye irritation) by the STE test method would require additional testing to
establish a definitive classification. Furthermore, the appropriate regulatory authorities
should be consulted before using the STE in a bottom-up approach under classification
schemes other than the UN GHS. The choice of the most appropriate test method and the
use of this Test Guideline should be seen in the context of the OECD Guidance Document
on an Integrated Approaches on Testing and Assessment for Serious Eye Damage and Eye
irritation (14).
3. The purpose of this test guideline (TG) is to describe the procedures used to
evaluate the eye hazard potential of a test chemical based on its ability to induce
cytotoxicity in the Short Time Exposure Test method. The cytotoxic effect of chemicals on
corneal epithelial cells is an important mode of action (MOA) leading to corneal epithelium
damage and eye irritation. Cell viability in the STE test method is assessed by the

© OECD, (2020)
You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.
2 491 OECD/OCDE
quantitative measurement, after extraction from cells, of blue formazan salt produced by
the living cells by enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide), also known as Thiazolyl Blue Tetrazolium Bromide
(3). The obtained cell viability after 5 minutes exposure is compared to the solvent control
(relative viability) and used to estimate the potential eye hazard of the test chemical. A test
chemical is classified as UN GHS Category 1 when both the 5% and 0.05% concentrations
result in a cell viability smaller than or equal to (≤) 70%. Conversely, a chemical is
predicted as UN GHS No Category when both 5% and 0.05% concentrations result in a cell
viability higher than (>) 70%.
4. The term “test chemical” is used in this Test Guideline to refer to what is tested and
is not related to the applicability of the STE test method to the testing of substances and/or
mixtures. Definitions are provided in Annex I.

INITIAL CONSIDERATIONS AND LIMITATIONS

5. This Test Guideline is based on a protocol developed by Kao Corporation (4),

which was the subject of two different validation studies: one by the Validation Committee
of the Japanese Society for Alternative to Animal Experiments (JSAAE) (5) and another
by the Japanese Center for the Validation of Alternative Methods (JaCVAM) (6). A peer
review was conducted by NICEATM/ICCVAM based on the validation study reports and
background review documents on the test method (7).
6. When used to identify chemicals (substances and mixtures) inducing serious eye
damage (UN GHS Category 1 (1), data obtained with the STE test method on 125 chemicals
(including both substances and mixtures), showed an overall accuracy of 83% (104/125), a
false positive rate of 1% (1/86), and a false negative rate of 51% (20/39) as compared to
the in vivo rabbit eye test (7). The false negative rate obtained is not critical in the present
context, since all test chemicals that induce a cell viability of ≤ 70% at a 5% concentration
and > 70% at 0.05% concentration (see Table 2: Prediction model below) would be
subsequently tested with other adequately validated in vitro test methods or, as a last option,
in the in vivo rabbit eye test, depending on regulatory requirements, and in accordance with
the sequential testing strategy and weight-of-evidence approaches currently recommended
(1) (8). Mainly mono-constituent substances were tested, although a limited amount of data
also exist on the testing of mixtures. The test method is nevertheless technically applicable
to the testing of multi-constituent substances and mixtures. When considering testing of
mixtures, difficult-to-test chemicals (e.g. unstable), or test chemicals not clearly within the
applicability domain described in this Guideline, upfront consideration should be given to
whether the results of such testing will yield results that are meaningful scientifically. The
STE test method showed no other specific shortcomings when used to identify test
chemicals as UN GHS Category 1. Investigators could consider using this test method on
test chemicals, whereby cell viability ≤ 70% at both 5% and 0.05% concentration should
be accepted as indicative of a response inducing serious eye damage that should be
classified as UN GHS Category 1 without further testing.
7. When used to identify chemicals (substances and mixtures) not requiring
classification for eye irritation and serious eye damage (i.e. UN GHS No Category), data
obtained with the STE test method on 130 chemicals (including both substances and
mixtures), showed an overall accuracy of 85% (110/130), a false negative rate of 12%
(9/73), and a false positive rate of 19% (11/57) as compared to the in vivo rabbit eye test
(7). If highly volatile substances (i.e. measured vapour pressure > 6kPa) and solid
substances other than surfactants are excluded from the dataset, the overall accuracy

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improves to 90% (92/102), the false negative rate to 2% (1/54), and the false positive to
19% (9/48) (7). Further work demonstrated that highly volatile substances can be correctly
tested when using mineral oil instead of saline as a solvent (15). The accuracy of the STE
test for highly volatile substances (i.e. vapour pressure > 6kPa) was then 95% (19/20), the
false negative rate was 0% (0/7), and the false positive rate was 8% (1/13). As a
consequence, the potential shortcoming of the STE test method when used to identify test
chemicals not requiring classification for eye irritation and serious eye damage (UN GHS
No Category) is a high false negative rate for solid chemicals (substances and mixtures)
other than surfactants and mixtures composed only of surfactants. Such chemicals are
excluded from the applicability domain of the STE test method (7). To that extent possible,
test chemicals that are sensitive to hydrolysis should be evaluated under conditions that do
not promote hydrolysis in order to avoid possible false negative results.
8. In addition to the chemicals mentioned in paragraphs 6 and 7, the STE test method
generated dataset also contains in-house data on 40 mixtures, which when compared to the
in vivo Draize eye test, showed an accuracy of 88% (35/40), a false positive rate of 50%
(5/10), and a false negative rate of 0% (0/30) for predicting mixtures that do not require
classification under the UN GHS classification system (9). The STE test method can
therefore be applied to identify mixtures as UN GHS No Category in a bottom-up approach
with the exception of solid mixtures other than those composed only of surfactants as an
extension of its limitation to solid substances. Furthermore, mixtures containing substances
with vapour pressure higher than 6kPa that do not dissolve in mineral oil, or that do not
form stable suspensions for at least 5 minutes, are not currently within the applicability
domain of the test method and may result in false negative outcomes.
9. The STE test method cannot be used for the identification of test chemicals as UN
GHS Category 2, Category 2A (eye irritation) or UN GHS Category 2B (mild eye
irritation), due to the considerable number of UN GHS Category 1 chemicals under-
predicted as UN GHS Category 2, 2A, or 2B and UN GHS No Category chemicals over-
predicted as UN GHS Category 2, 2A, or 2B (7). For this purpose, further testing with
another suitable method may be required.
10. The STE test method is suitable for test chemicals that are dissolved or uniformly
suspended for at least 5 minutes in physiological saline, 5% dimethyl sulfoxide (DMSO)
in saline, or mineral oil (see paragraph 17 for solvent choice). The STE test method is not
suitable for test chemicals that are insoluble or cannot be uniformly suspended for at least
5 minutes in physiological saline, 5% DMSO in saline, or mineral oil. The use of mineral
oil in the STE test method is possible because of the short-time exposure. Therefore, the
STE test method is suitable for predicting the eye hazard potential of water-insoluble test
chemicals (e.g., long-chain fatty alcohols or ketones) provided that they are miscible in at
least one of the three above proposed solvents (4).

PRINCIPLE OF THE TEST

11. The STE test method is a cytotoxicity-based in vitro assay that is performed on a
confluent monolayer of Statens Seruminstitut Rabbit Cornea (SIRC) cells, cultured on a
96-well polycarbonate microplate (4). After five-minute exposure to both a 5% and a 0,05%
concentration of a test chemical, the cytotoxicity is quantitatively measured as the relative
viability of SIRC cells using the MTT assay (4). Decreased cell viability is used to predict
potential adverse effects leading to ocular damage.

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12. It has been reported that 80% of a solution dropped into the eye of a rabbit is
excreted through the conjunctival sac within three to four minutes, while greater than 80%
of a solution dropped into the human eye is excreted within one to two minutes (10). The
STE test method attempts to approximate these exposure times and makes use of
cytotoxicity as an endpoint to assess the extent of damage to SIRC cells following a five-
minute exposure to the test chemical.

DEMONSTRATION OF PROFICIENCY

13. Prior to routine use of the STE test method described in this test guideline,
laboratories should demonstrate technical proficiency by correctly classifying the eleven
substances recommended in Table 1. These substances were selected to represent the full
range of responses for serious eye damage or eye irritation based on results of in vivo rabbit
eye tests (TG 405) and the UN GHS classification system (1). Other selection criteria
included that the substances should be commercially available, that high-quality in vivo
reference data should be available, and that high quality in vitro data from the STE test
method should be available (3). In situations where a listed substance is unavailable or
where justifiable, another substance for which adequate in vivo and in vitro reference data
are available could be used provided that the same criteria as described here are used.

Table 1: List of Proficiency Substances

Physic STE UN GHS
In Vivo UN Solvent in
Substance CASRN Chemical class1 al Cat.
GHS Cat.2 STE test
state
Benzalkonium
8001-
chloride Onium compound Liquid Category 1 Saline Category 1
54-5
(10%, aqueous)
Triton X-100 9002-
Ether Liquid Category 1 Saline Category 1
(100%) 93-1
Heterocyclic
18472- compound; Bromine
Acid Red 92 compound; Chlorine
Solid Category 1 Saline Category 1
87-2
compound

Sodium 1310- Alkali; Inorganic

chemical
Solid Category 13 Saline Category 1
hydroxide 73-2
No stand-
Lactone;
alone
Butyrolactone 96-48-0 Heterocyclic Liquid Category 2A Saline
compound prediction can
be made
No stand-
111-87- Mineral alone
1-Octanol Alcohol Liquid Category 2A/B4
5 Oil prediction can
be made
Alcohol; No stand-
Cyclopentanol 96-41-3 Hydrocarbon, cyclic
Liquid Category 2A/B5 Saline
alone

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 OECD/OCDE 491  5
prediction can
be made

2-Ethoxyethyl 111-15-
Alcohol; Ether Liquid No Category Saline No Category
acetate 9
112-40- Hydrocarbon, Mineral
Dodecane acyclic Liquid No Category No Category
3 Oil
Methyl isobutyl 108-10- Mineral
Ketone Liquid No Category No Category
ketone 1 Oil
Glycerol 56-81-5
Alcohol Liquid No Category Saline No Category

Abbreviations: CAS RN = Chemical Abstracts Service Registry Number

1
Chemical classes were assigned using information obtained from previous NICEATM
publications and if not available, using the National Library of Medicine's Medical Subject
Headings (MeSH®) (via ChemIDplus® [National Library of Medicine], available at
http://chem.sis.nlm.nih.gov/chemidplus/) and structure determinations made by
NICEATM.
2
Based on results from the in vivo rabbit eye test (OECD TG 405) and using the UN GHS
(1).
3
Classification as Cat.1 is based on skin corrosive potential of 100% sodium hydroxide
(listed as a proficiency chemical with skin corrosive potential in OECD TG 435) and the
criterion for UN GHS category 1 (1).
4
Classification as 2A or 2B depends on the interpretation of the UN GHS criterion for
distinguishing between these two categories, i.e., 2 out of 6 vs 4 out of 6 animals with
effects at day 7 necessary to generate a Category 2A classification. The in vivo dataset
included 2 studies with 3 animals each. In one study two out of three animals showed effects
at day 7 warranting a Cat. 2A classification (11), whereas in the second study all endpoints
in all three animals recovered to a score of zero by day 7 warranting a Cat. 2B classification
(12).
5
Classification as 2A or 2B depends on the interpretation of the UN GHS criterion for
distinguishing between these two categories, i.e., 1 out of 3 vs 2 out of 3 animals with
effects at day 7 necessary to generate a Category 2A classification. The in vivo study
included 3 animals. All endpoints apart from corneal opacity and conjunctivae redness in
one animal recovered to a score of zero by day 7 or earlier. The one animal that did not
fully recover by day 7 had a corneal opacity score of 1 and a conjunctivae redness of 1 (at
day 7) that fully recovered at day 14 (11).

PROCEDURE

Preparation of the Cellular Monolayer

14. The rabbit cornea cell line, SIRC should be used for performing the STE test
method. It is recommended that SIRC cells are obtained from a well-qualified cell bank,
such as American Type Culture Collection CCL60.

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15. SIRC cells are cultured at 37°C under 5% CO2 and humidified atmosphere in a
culture flask containing a culture medium comprising Eagle's minimum essential medium
(MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50–100
units/mL penicillin and 50–100 µg/mL streptomycin. Cells that have become confluent in
the culture flask should be separated using trypsin-ethylenediaminetetraacetic acid
solution, with or without the use of a cell scraper. Cells are propagated (e.g. 2 to 3 passages)
in a culture flask before being employed for routine testing, and should undergo no more
than 25 passages from thawing.
16. Cells ready to be used for the STE test are then prepared at the appropriate density
and seeded into 96-well plates. The recommended cell seeding density is 6.0 × 103 cells
per well when cells are used four days after seeding, or 3.0 × 103 cells per well when cells
are used five days after seeding, at a culture volume of 200 µL. Cells used for the STE test
that are seeded in a culture medium at the appropriate density will reach a confluence of
more than 80% at the time of testing, i.e., four or five days after seeding.

Application of the Test Chemicals and Control Substances

17. The first choice of solvent for dissolving or suspending test chemicals is
physiological saline. If the test chemical demonstrates low solubility or cannot be dissolved
or suspended uniformly for at least five minutes in saline, 5% DMSO (CAS#67-68-5) in
saline is used as a second choice solvent. For test chemicals that cannot be dissolved or
suspended uniformly for at least five minutes in either saline or 5% DMSO in saline,
mineral oil (CAS#8042-47-5) is used as a third choice solvent. For highly volatile test
chemicals (i.e. vapor pressure over 6 kPa) mineral oil is used as a solvent, provided the test
chemical dissolves or forms a stable suspension for at least five minutes in mineral oil.
18. Test chemicals are dissolved or suspended uniformly in the selected solvent at 5%
(w/w) concentration and further diluted by serial 10-fold dilution to 0.5% and 0.05%
concentration. Each test chemical is to be tested at both 5% and 0.05% concentrations.
Cells cultured in the 96-well plate are exposed to 200 µL/well of either a 5% or a 0.05%
concentration of the test chemical solution (or suspension), for five minutes at room
temperature. Test chemicals (mono-constituent substances or multi-constituent substances
or mixtures) are considered as neat substances and diluted or suspended according to the
method, regardless of their purity.
19. The culture medium described in paragraph 15 is used as a medium control in each
plate of each repetition. Furthermore, cells are to be exposed also to solvent control samples
in each plate of each repetition. The solvents listed in paragraph 17 have been confirmed
to have no adverse effects on the viability of SIRC cells.
20. In the STE test method, 0.01% Sodium lauryl sulfate (SLS) in saline is to be used
as a positive control in each plate of each repetition. In order to calculate cell viability of
the positive control, each plate of each repetition has to also include a saline solvent control.
21. A blank is necessary to determine compensation for optical density and should be
performed on wells containing culture medium or phosphate buffered saline, but no
calcium and magnesium (PBS-) or cells.
22. Each sample (test chemical at 5% and 0.05%, medium control, solvent control, and
positive control) should be tested in triplicate in each repetition by exposing the cells to
200 µL of the appropriate test or control chemical for five minutes at room temperature.

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23. Benchmark substances are useful for evaluating the ocular irritancy potential of
unknown chemicals of a specific chemical or product class, or for evaluating the relative
irritancy potential of an ocular irritant within a specific range of irritant responses.

Cell Viability Measurement

24. After exposure, cells are washed twice with 200 μL of PBS and 200 μL of MTT
solution (0.5 mg MTT/mL of culture medium) is added. After a two-hour reaction time in
an incubator (37˚C, 5% CO2), the MTT solution is decanted, MTT formazan is extracted
by adding 200 μL of 0.04 N hydrochloric acid-isopropanol for 60 minutes in the dark at
room temperature, and the absorbance of the MTT formazan solution is measured at 570
nm with a plate reader. Interference of test chemicals with the MTT assay (by colorants or
direct MTT reducers) only occurs if significant amount of test chemical is retained in the
test system following rinsing after exposure. While this is often the case for the 3D
reconstructed human cornea or Reconstructed human epidermis, it is less likely to occur in
the 2D cell cultures used for the STE test method. However, because residual material from
colorants or direct MTT reducers could interfere with the measurement of optical density,
STE users should evaluate such results with caution. If the test results in a Category 1
prediction, then no further actions to address potential interference are needed. Where
possible, data should be generated to determine whether such interference is occurring (e.g.,
conducting an experiment to compare MTT assay OD measurements from test article-
treated wells containing SIRC cells in comparison to test article-treated wells containing
no cells). If MTT interference is expected to affect the results, alternative cytotoxicity
assays (e.g. neutral red) can be used as long as it can be shown to provide similar results as
MTT assay, e.g. by testing the proficiency substances in Table 1, and if historical data are
available to derive comparable run acceptance criteria (see paragraph 29).

Interpretation of Results and Prediction Model

25. The optical density (OD) values obtained for each test chemical are then used to
calculate cell viability relative to the solvent control, which is set at 100%. The relative cell
viability is expressed as a percentage and obtained by dividing the OD of test chemical by
the OD of the solvent control after subtracting the OD of blank from both values.

(OD570 of test chemical) – (OD570 of blank)

Cell viability (%) = × 100
(OD570 of solvent control) – (OD570 of blank)

Similarly, the relative cell viability of each solvent control is expressed as a percentage and
obtained by dividing the OD of each solvent control by the OD of the medium control after
subtracting the OD of blank from both values.
26. Three independent repetitions, each containing three replicate wells (i.e., n=9),
should be performed. The arithmetic mean of the three wells for each test chemical and
solvent control in each independent repetition is used to calculate the arithmetic mean of
relative cell viability. The final arithmetic mean of the cell viability is calculated from the
three independent repetitions.
27. The cell viability cut-off values for identifying test chemicals inducing serious eye
damage (UN GHS Category 1) and test chemicals not requiring classification for eye
irritation or serious eye damage (UN GHS No Category) are given hereafter.

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Table 2: Prediction model of the STE test method

Cell viability
UN GHS
Applicability
Classification
At 5% At 0.05%

Substances and mixtures1, with the

exception of
> 70% > 70% No Category solid chemicals (substances and mixtures)
other than surfactants and mixtures
composed only of surfactants
No stand-alone
≤ 70% > 70% prediction can be Not applicable
made2
≤ 70% ≤ 70% Category 1 Substances and mixtures 3
1
Mixtures containing test chemicals with vapour pressure higher than 6kPa and that do not either
dissolve or form a stable suspension in mineral oil are currently not within the applicability domain
of the test method and can generate under-predictions.
2
No stand-alone prediction can be made from this result in isolation. The result of the STE test
should be considered in the context of an IATA (14) for classification purposes.
3
Based on results obtained mainly with mono-constituent substances, although a limited amount of
data also exist on the testing of mixtures. The test method is nevertheless technically applicable to
the testing of multi-constituent substances and mixtures. Before use of this Test Guideline on a
mixture for generating data for an intended regulatory purpose, it should be considered whether, and
if so why, it may provide adequate results for that purpose. Such considerations are not needed,
when there is a regulatory requirement for testing of the mixture.

Acceptance Criteria
28. Test results are judged to be acceptable when the following criteria are all satisfied:
a) Optical density of the medium control (exposed to culture medium) should
be 0.3 or higher after subtraction of blank optical density.
b) Viability of the solvent control should be 80% or higher relative to the
medium control. If multiple solvent controls are used in each repetition, each of
those controls should show cell viability greater than 80% to qualify the test
chemicals tested with those solvents.
c) The cell viability obtained with the positive control (0.01% SLS) should be
within two standard deviations of the historical mean. The upper and lower
acceptance boundaries for the positive control should be frequently updated i.e.,
every three months, or each time an acceptable test is conducted in laboratories
where tests are conducted infrequently (i.e., less than once a month). Where a
laboratory does not complete a sufficient number of experiments to establish a
statistically robust positive control distribution, it is acceptable that the upper and
lower acceptance boundaries established by the method developer are used, i.e.,

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between 21.1% and 62.3% according to its laboratory historical data, while an
internal distribution is built during the first routine tests.
If any of the above criteria a), b) or c) are not met, an additional repetition should
be performed.
d) Standard deviation of the final cell viability derived from three independent
repetitions should be less than 15% for both 5% and 0.05% concentrations of the
test chemical. If the standard deviation is greater than or equal to 15%, the results
should not be used and three more repetitions should be performed.

DATA AND REPORTING

Data
29. Data for each individual well (e.g., cell viability values) of each repetition as well
as overall mean, standard deviation (SD), and classification are to be reported.

Test Report
30. The test report should include the following information:

Test Chemical and Control Substances

 Mono-constituent substance: chemical identification, such as IUPAC or CAS
name(s), CAS registry number(s), SMILES or InChI code, structural formula,
and/or other identifiers;
 Multi-constituent substance, UVCB and mixture: Characterization as far as
possible by e.g., chemical identity (see above), purity, quantitative occurrence and
relevant physicochemical properties (see above) of the constituents, to the extent
available;
 Physical state, volatility, pH, LogP, molecular weight, chemical class, and
additional relevant physicochemical properties relevant to the conduct of the study,
to the extent available;
 Purity, chemical identity of impurities as appropriate and practically feasible, etc;
 Treatment prior to testing, if applicable (e.g., warming, grinding);
 Storage conditions and stability to the extent available;
 Solubility for at least five minutes in a selected solvent (e.g. dissolution or stable
suspension).

Test Method Conditions and Procedures

 Name and address of the sponsor, test facility and study director;
 Description of the test method used;
 Cell line used, its source, passage number and confluence of cells used for testing;
 Details of test procedure used;
 Number of repetitions and replicates used;

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 Test chemical concentrations used (if different than the ones recommended);
 Justification for choice of solvent for each test chemical;
 Duration of exposure to the test chemical (if different than the one recommended);
 Description of any modifications of the test procedure;
 Description of evaluation and decision criteria used;
 Reference to historical positive control mean and Standard Deviation (SD):
 Demonstration of proficiency of the laboratory in performing the test method (e.g.
by testing of proficiency substances) or demonstration of reproducible performance
of the test method over time.

Results
 For each test chemical and control substance, and each tested concentration,
tabulation should be given for the individual OD values per replicate well, the
arithmetic mean OD values for each independent repetition, the % cell viability for
each independent repetition, and the final arithmetic mean % cell viability and SD
over the three repetitions;
 Results for the medium, solvent and positive control demonstrating suitable study
acceptance criteria;
 Description of other effects observed, including retainment of significant amounts
of coloured and/or direct MTT reducer test chemical following rinsing after
exposure;
 The overall derived classification with reference to the prediction model/decision
criteria used.

Discussion of the Results

Conclusions

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LITERATURE

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Safety Publications,Series on Testing and Assessment (No. 34.). Organisation for
Economic Cooperation and Development, Paris.
14) OECD (2018). Guidance Document on an Integrated Approaches on Testing and
Assessment for Serious Eye Damage and Eye irritation. Series on Testing and Assessment
No.263. ENV Publications, Organisation for Economic Cooperation and Development,
Paris.
15) Abo T, et al. (2018). Expansion of the applicability domain for highly volatile
substances on the Short Time Exposure test method and the predictive performance in
assessing eye irritation potential. J. Toxicol. Sci., 43, 407-422

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ANNEX I

DEFINITIONS

Accuracy: The closeness of agreement between test method results and accepted reference
values. It is a measure of test method performance and one aspect of relevance. The term
is often used interchangeably with concordance to mean the proportion of correct outcomes
of a test method (13).
Benchmark substance: A substance used as a standard for comparison to a test chemical.
A benchmark substance should have the following properties; (i) a consistent and reliable
source(s); (ii) structural and functional similarity to the class of substances being tested;
(iii) known physical/chemical characteristics; (iv) supporting data on known effects, and
(v) known potency in the range of the desired response.
Bottom-Up Approach: A step-wise approach used for a test chemical suspected of not
requiring classification for eye irritation or serious eye damage, which starts with the
determination of chemicals not requiring classification (negative outcome) from other
chemicals (positive outcome)
Chemical: means a substance or mixture.
Eye irritation: Production of change in the eye following the application of a test chemical
to the anterior surface of the eye, which are fully reversible within 21 days of application.
Interchangeable with “reversible effects on the eye” and with UN GHS Category 2 (1)
False negative rate: The proportion of all positive chemicals falsely identified by a test
method as negative. It is one indicator of test method performance.
False positive rate: The proportion of all negative chemicals that are falsely identified by
a test method as positive. It is one indicator of test method performance.
Hazard: Inherent property of an agent or situation having the potential to cause adverse
effects when an organism, system or (sub) population is exposed to that agent.
Medium control: An untreated replicate containing all components of a test system. This
sample is processed with test chemical-treated samples and other control samples to
determine whether the solvent interacts with the test system.
Mixture: A mixture or a solution composed of two or more substances in which they do
not react (1).
Mono-constituent substance: A substance, defined by its quantitative composition, in
which one main constituent is present to at least 80% (w/w).
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue
tetrazolium bromide.
Multi-constituent substance: A substance, defined by its quantitative composition, in
which more than one main constituent is present in a concentration ≥ 10% (w/w) and <
80% (w/w). A multi-constituent substance is the result of a manufacturing process. The
difference between mixture and multi-constituent substance is that a mixture is obtained by
blending of two or more substances without chemical reaction. A multi-constituent
substance is the result of a chemical reaction.

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OD: Optical Density.
Positive control: A replicate containing all components of a test system and treated with a
substance known to induce a positive response. To ensure that variability in the positive
control response across time can be assessed, the magnitude of the positive response should
not be excessive.
Relevance: Description of relationship of the test to the effect of interest and whether it is
meaningful and useful for a particular purpose. It is the extent to which the test correctly
measures or predicts the biological effect of interest. Relevance incorporates consideration
of the accuracy (concordance) of a test method (10).
Reliability: Measures of the extent that a test method can be performed reproducibly within
and between laboratories over time, when performed using the same protocol. It is assessed
by calculating intra- and inter-laboratory reproducibility and intra-laboratory repeatability
(13).
Sensitivity: The proportion of all positive/active chemicals that are correctly classified by
the test. It is a measure of accuracy for a test method that produces categorical results, and
is an important consideration in assessing the relevance of a test method (10).
Serious eye damage: Production of tissue damage in the eye, or serious physical decay of
vision, following application of a test chemical to the anterior surface of the eye, which is
not fully reversible within 21 days of application. Interchangeable with “irreversible effects
on the eye” and with UN GHS Category 1 (1).
Solvent/vehicle control: An untreated sample containing all components of a test system,
including the solvent or vehicle that is processed with the test chemical-treated and other
control samples to establish the baseline response for the samples treated with the test
chemical dissolved in the same solvent or vehicle. When tested with a concurrent medium
control, this sample also demonstrates whether the solvent or vehicle interacts with the test
system.
Specificity: The proportion of all negative/inactive chemicals that are correctly classified
by the test. It is a measure of accuracy for a test method that produces categorical results
and is an important consideration in assessing the relevance of a test method (13).
Substance: Chemical elements and their compounds in the natural state or obtained by any
production process, inducing any additive necessary to preserve the stability of the product
and any impurities deriving from the process used, but excluding any solvent which may
be separated without affecting the stability of the substance or changing it composition (1).
Surfactant: Also called surface-active agent, this is a chemical such as a detergent, that
can reduce the surface tension of a liquid and thus allow it to foam or penetrate solids; it is
also known as a wetting agent.
Test chemical: The term "test chemical" is used to refer to what is being tested.
Tiered testing strategy: A stepwise testing strategy where all existing information on a
test chemical is reviewed, in a specified order, using a weight of evidence process at each
tier to determine if sufficient information is available for a hazard classification decision,
prior to progression to the next tier. If the irritancy potential of a test chemical can be
assigned based on the existing information, no additional testing is required. If the irritancy
potential of a test chemical cannot be assigned based on the existing information, a step-

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wise sequential animal testing procedure is performed until an unequivocal classification
can be made.
Top-Down Approach: step-wise approach used for a test chemical suspected of causing
serious eye damage, which starts with the determination of chemicals inducing serious eye
damage (positive outcome) from other chemicals (negative outcome).
United Nations Globally Harmonized System of Classification and Labelling of
Chemicals (UN GHS): A system proposing the classification of chemicals (substances and
mixtures) according to standardized types and levels of physical, health and environmental
hazards, and addressing corresponding communication elements, such as pictograms,
signal words, hazard statements, precautionary statements and safety data sheets, so that to
convey information on their adverse effects with a view to protect people (including
employers, workers, transporters, consumers and emergency responders) and the
environment (1).
UN GHS Category 1: See “Serious eye damage”.
UN GHS Category 2: See “Eye irritation”.
UN GHS No Category: Chemicals that are not classified as UN GHS Category 1 or 2 (2A
or 2B).
UVCB: substances of unknown or variable composition, complex reaction products or
biological materials.

©OECD 2020