[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Molecular formula:

C6H12N2 - C47H77O16N4

IUPAC Name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Test material form:

liquid

Details on test material:

Name of test item: Emuldur 3643
Test item No.: 19/0071-2
Batch identification: 27771924U0
Purity: 100 % (UVCB*, for details see analytical characterization No.20L00150) *Substances of unknown or variable composition, complex reaction products.
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: July 15, 2021
Storage conditions: Ambient (RT)
Physical state / color: Liquid / colorless to yellowish, clear

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Three-dimensional human cornea model
The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct
composed of normal human-derived epidermal keratinocytes used to model the human corneal
epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell
culture inserts (MILLICELLs, 10 mm diameter) and are available commercially as kits
(EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 34910 (Certificate of Analysis see Appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

ANALYSES
No analysis of the test-substance preparation was performed because the test substance was
applied undiluted.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

see details on study design

Duration of treatment / exposure:

see details on study design

Duration of post- treatment incubation (in vitro):

see details on study design

Number of animals or in vitro replicates:

2

Details on study design:

EXPERIMENTAL PROCEDURE
Pretest for direct MTT reduction
The direct reduction of MTT by a test substance interferes with the color density produced by
metabolic capacity of the tissue and would falsify the test results.
In order to assess the ability of the test material to reduce MTT directly, a pretest (experimental
conduct in accordance with GLP, but without a GLP status) was performed as described below.
The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark
at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently.
If the color of the MTT solution or (in case of water-insoluble test substances) the border to the
water phase turned blue / purple, the test substance was presumed to reduce MTT directly.
In case of direct MTT reduction, two freeze-killed control tissues (KC) were treated additionally
with each the test article and the negative control in the same way as described in section 3.6.
Based on the result of the pretest, it was judged that application of killed control tissues is
necessary.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 129) using the
same control tissues (NC and PC).
Two tissues were treated with each the test substance, the PC and the NC.
In addition, two killed tissues were used for each the test substance and the NC to detect direct
MTT reduction.
There are two separate protocols for liquids and solids differing in exposure time and postincubation
period. Due to the physical condition of the test substance, the protocol for liquids
was applied.

Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with
1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the
pre-incubation medium was replaced with fresh medium, and preconditioning continued in the
incubator at standard culture conditions for 16 – 24 hours.

Pretreatment of the tissues
After pre-incubation, the tissues were pretreated with 20 μL PBS in order to wet the tissue
surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance
Using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering
the whole tissue surface.
Control tissues were applied concurrently with 50 μL sterile deionized water (NC, NC KC) or
with 50 μL methyl acetate (PC) or test substance (KC).
After application, the tissues were placed into the incubator until the total exposure time of
30 minutes was completed.

Removal of the test substance and postincubation period
In order to remove the test substance, the tissues were washed with sterile PBS. For this
purpose, the tissues were immersed and swiveled three times in each of three beakers filled
with PBS. Washed tissues were immersed immediately into 12-well plates pre-filled with
5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance.
After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and
transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation
period).

MTT incubation
After the post-incubation period, the assay medium was replaced with 0.3 mL MTT solution,
and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of
the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts
was spectrophotometrically determined. Blank values were established of 4 microtiter wells
filled with isopropanol for each microtiter plate.

Data evaluation
Table(s) of measured parameters that are presented in the report were produced using a
computer-based tabular calculation software. The mean and individual data were not always
rounded, but the significant digits were produced by changing the display format. As a
consequence, calculation of mean values by using the individual data presented in the report
will in some instances yield minor variations in value.
Principle
The OD570 values determined for the various tissues are a
measure of their viability. The ratio of the OD570 of tissues
treated with the test material and the mean OD570 values of the
NC (percent of control) is used for evaluating whether a test
material was an irritant.

Calculation of individual and mean optical densities
The corrected measured OD570 value for each individual tissue
was calculated by subtracting the mean blank value of the
respective microtiter plate from the respective individual tissue
OD570 value. The mean OD570 for a test group of two tissues
treated in the same way was calculated.

Application of measurements using killed control tissues
In case of direct MTT reduction by the test substance, the OD570
values measured in the freeze-killed control tissues (KC) will be
used to correct the mean OD570 of the tissues treated with the
test substance (mean corrected OD570 KC). Since a killed tissue
might still have a residual enzyme activity that is able to produce
some formazan net, OD570 KC is calculated by subtracting the
OD570 KC of the NC from the OD570 KC of the test substance.
In case the net OD570 KC is greater than zero, it is subtracted
from the respective mean OD570 to result in the mean corrected
OD570 KC. The mean corrected OD570 KC represents the
formazan production linked to the tissue viability and therefore
indicates the cytotoxic potency of the test substance.

Tissue viability
The quantification of tissue viability is presented as ratio of the
mean OD570 (or mean corrected OD570 KC if applicable) divided
by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
In case one of the acceptance criteria below was not met, repetition of the test was considered.
Barrier function and Quality control (QC)
The supplier demonstrates that each batch of the model used
meets the defined production release criteria. MatTek
determines the ET50 (min) value following exposure to 100 μL
of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200)
batch. The ET50 must fall within an established range based on
a historical database of results.
The following acceptability range (upper and lower limit) for the
ET50 is established by the supplier as described in the cited
OECD Guideline.
Lower acceptance limit: ET50 = 12.2 min
Upper acceptance limit: ET50 = 37.5 min
EpiOcular QC (OCL-200) batch release see Appendix

Acceptance criteria for the NC
The absolute OD570 of the NC tissues in the MTT test is an
indicator of tissue viability obtained in the testing laboratory
after the shipping and storing procedure and under specific
conditions of the assay. Tissue viability is acceptable if the
mean OD570 of the NC is  0.8. The mean OD570 of the NC
should not exceed 2.8.

Acceptance criteria for the PC
Methyl acetate used as PC usually leads to a tissue viability of
approx. 25%. A viability of < 50% is acceptable.

Acceptance criteria for tissue variability
Two tissues were treated under the same conditions.
A variability between the tissues is considered to be acceptable
if the relative difference of the viability is < 20%.

Acceptance criteria for the KC
The OD570 of the killed control tissues treated as negative
control should be <= 0.35. The value for direct MTT reduction of
a test substance should be <= 30% of the NC.

EVALUATION OF RESULTS
The eye irritation potential of the test material is predicted from the mean relative tissue
viabilities compared to the negative control tissues treated concurrently with sterile water. A
chemical is considered as "non-irritant” (no UN GHS Category) if the mean relative tissue
viability with a test material is greater than 60%.
A single test composed of at least two tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in case of borderline results such as non-concordant
replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off value,
a second test should be considered as well as a third one in case of discordant results between
the first two tests.
The following decision criteria apply:
Mean tissue viability (% of negative control)
<= 60 % No prediction can be made
> 60 % No UN GHS Category

A “borderline“ evaluation (60 ± 5%) was determined statistically using historic BASF data and
hence considers the variance of the test method. This evaluation is confirming the borderline
range provided in OECD Guideline 492.

HISTORIC CONTROL DATA
Historic control values of negative and positive controls collected over an appropriate period
are presented in section 4.2. These data demonstrate the reproducibility of results and
robustness of the procedures. They are used to derive suitable acceptance criteria (see
section 3.7.) for the test system.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Run / experiment:

negative control viable tissue

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

mean percent tissue viability 

Run / experiment:

negative control freeze-killed control tissues

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 3

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Test substance viable tissue

Value:

13.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Test substance freeze-killed tissue

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 5

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Final relative mean viability of tissues after KC correction [% of NC]:

Value:

10.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 6

Irritation parameter:

mean percent tissue viability 

Run / experiment:

positive control viable tissue

Value:

35.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in
parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean
viability 3.0% of NC). Thus, for the test substance the final relative mean viability is given after
KC correction.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results observed and by applying the evaluation criteria described in chapter 3.8., it was concluded that Emuldur 3643 shows an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen.
The test method does not allow for the evaluation of serious eye damage. The result does not exclude a serious eye irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.