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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 October 2017 to 28 November 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
GLP study conducted in compliance with OECD Guideline No. 492 without any deviation.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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)
Version / remarks:
(Adopted 28 July 2015)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Storage condition of test material: Stored at room temperature, protected from light

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
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.

Assay medium: OCL-200-ASY/Assay Medium
MTT diluent: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ and OCL-200-ASY/Assay Medium used for diluting MTT
Pre-treatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+
Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT), 1.0 mg / mL MTT diluent
Extracting agent: 2-propanol

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 50 mg

- Control tissues were concurrently applied with 50 μL of sterile distilled water (NC) or with 50 μL of methyl acetate (PC).
Duration of treatment / exposure:
6 hours ± 15 minutes
Duration of post- treatment incubation (in vitro):
18 hours ± 15 minutes
Number of animals or in vitro replicates:
Details on study design:

To assess the ability of the test material to directly reduce MTT a pretest was performed. Fifty milligrams of the test substance was added to 1 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 180 minutes. As a result, the change in color was not observed. However, the test substance was changed color in to light purple and it was judged that the test substance had reactivity with MTT. Therefore, interference of the test substance with MTT (interference test) was conducted in the eye irritation test.
-Basic procedure:
Two tissues were treated with each, the test substance, the PC and the NC.
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 60 ± 5 minutes the preincubation 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 the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
-Application of the test substance:
Fifty milligrams of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile distilled water (NC) or with 50 μL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours ± 15 minutes was completed.
-Removal of the test substance and postincubation period:
To remove the test substance, the tissues were washed with sterile PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium in order to remove residual test substance. After 25 ± 2 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 ± 15 minutes.
-MTT incubation:
After the post-incubation period, the assay medium was replaced by 0.3 mL/well MTT solution and the tissues were incubated in the incubator for 180 ± 10 minutes.
After incubation, the outside of tissue inserts was wiped. All tissues were transferred into new 6-well plates filled with 2 mL/well of 2-propanol. The plate was put into a plastic bag, and extraction was performed at room temperature for 2 hours or more using a plate shaker. The extracts were mixed to obtain homogeneous solutions.
-Optical Density measurements:
The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 8 microtiter wells filled with 2-propanol in a microtiter plate.
- RhCE tissue construct used, including batch number:
Tissue model: OCL-200
Tissue Lot Number: 20994
Supplier: MatTek Corporation

- Evaluation of results:
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile distilled water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.

Results and discussion

In vitro

Irritation parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
6 hours
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria
See section 13.2 for classification justification
Based on the observed results and applying the evaluation criteria it was concluded, that White vanilla shows a serious eye damage or an eye irritation potential in the EpiOcularTM in vitro eye irritation test under the test conditions chosen.