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Toxicological information

Eye irritation

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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:
other: Well documented and reliable GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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 (incl. QA statement)
Remarks:
BASF SE, Ludwigshafen, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethoxyethyl 2-cyano-2-[(1Z)-3-[(3-methoxypropyl)amino]cyclohex-2-en-1-ylidene]acetate
EC Number:
700-860-3
Cas Number:
1419401-88-9
Molecular formula:
C17 H26 N2 O4
IUPAC Name:
2-ethoxyethyl 2-cyano-2-[(1Z)-3-[(3-methoxypropyl)amino]cyclohex-2-en-1-ylidene]acetate

Test animals / tissue source

Species:
other: human derived epidermal keratinozytes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
The EpiOcular[TM] model is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcular[TM] tissues (surface area 0.6 cm²) are cultured on specially prepared cell culture inserts and are commercially available as kits, containing 24 tissues on shipping agarose.

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
and 3% (w/w) in deionized water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied
1st dose group: ca. 50 mg test material
2nd dose group: bulk volume of ca. 50 μL test material (corresponding to ca. 8 mg per tissue)
3rd dose group: 50 μL of a 3% (w/w) test substance suspension in deionized water

- Concentration (if solution): undiluted, 3% (w/w) in deionized water
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours post incubation
Number of animals or in vitro replicates:
2 tissues
Details on study design:
Details of the test procedure used
- Basic procedure: Several test substances were tested in parallel within the present test using the same control tissues (NC and PC). Two tissues were treated with each the test substance (per dose group), the PC and the NC. There are two separate protocols for liquids and solids differing in exposure time and postincubation period. Due to the physical condition of the undiluted test substance, the protocol for solids was applied for all dose groups.
- 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 - 34 minutes.
- Application of the test substance: 3 dose groups were tested as follows:
- 1st dose group: ca. 50 mg test material per tissue applied with a syringe with head cut off.
- 2nd dose group: a bulk volume of ca. 50 μL test material applied with a sharp spoon (corresponding to ca. 8 mg per tissue), the bulk volume is covering the whole tissue surface.
- 3rd dose group: 50 μL of a 3% (w/w) test substance suspension in deionized water applied using a positive displacement pipette.
Control tissues were applied concurrently with 50 μL sterile deionized water (NC) or with 50 μL methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.
- Removal of the test substance and postincubation period: 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 25 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 18 hours (postincubation period).
- MTT incubation: 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 produced metabolically by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

RhCE tissue construct used, including batch number
- EpiOcularTM model (OCL-200)
- Tissue Lot Number: 30609
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Incubation conditions during exposure, and post-exposure incubation periods:
- 37°C ± 1°C, 5% ± 1%CO2, 90% ± 10% relative humidity

Indication of controls used for direct MTT-reducers and/or colouring test chemicals
- 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. 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. Based on the results of a pretest with the 3% aqueous dilution and a previous pretest (Project number 62V0473/11A564) performed with the undiluted test substance, it was judged that application of killed control tissues (KC) is not necessary.

Wavelength used for quantifying MTT formazan
- Measurement using a filter wavelength 570 nm without reference filter; Spectrophotometer: SunriseTM Absorbance Reader.

Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
- Decision criteria:
- Mean tissue viabiltiy (% of negative control) > 60 % -> No UN GHS Category
- Mean tissue viabiltiy (% of negative control) <= 60 % -> No prediction can be made. Further testing with other test methods will be required because the EpiOcular EIT shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
The eye irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as eye irritant if the mean relative tissue viability with a test material is less than or equal to 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.
A “borderline“ evaluation (60 ± 5%) was determined statistically using historic data of the testing laboratory and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 492.
- 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
- 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 2.5.
- 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%.

Historical control data
Historic control values of negative and positive controls collected over an appropriate period, demonstrate the reproducibility of results and robustness of the procedures. They are used to derive the acceptance criteria for the test system.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: viability [% of NC]
Remarks:
mean of 2 tissues
Run / experiment:
1st dose group (ca. 50 mg)
Value:
93.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Inter-tissue variability [%] 13.5
Irritation parameter:
other: viability [% of NC]
Remarks:
mean of 2 tissues
Run / experiment:
2nd dose group (bulk volume 50 μL)
Value:
106.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Inter-tissue variability [%] 2.9
Irritation parameter:
other: viability [% of NC]
Remarks:
mean of 2 tissues
Run / experiment:
3rd dose group (3% aqueous suspension)
Value:
103.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Inter-tissue variability [%] 12.3
Other effects / acceptance of results:
The test substance is not able to reduce MTT directly, which was demonstrated in pretests. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.
Slight or moderate compound residues (light yellowish colored) remained on the tissues treated with 8 mg (2nd dose group) or 50 mg (1st dose group) test substance after the washing procedure. However, this did not interfere with the MTT assay as the test substance was not able to reduce MTT directly which was demonstrated in a pretest. In order to avoid contamination of the extract solution with the compound residues, the tissues treated with the test substance were extracted only from below without piercing.
Application of the positive control methyl acetate showed a relative mean viability of the tissues of 21.9% and reflects the expected sensitivity of the tissues.
The mean OD570 of the negative control (deionized water) fulfill the acceptance criteria and demonstrate the validity of the assay.

Applicant's summary and conclusion