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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:
The study was conducted between 26 April 2017 and 12 May 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
GLP compliance:
yes (incl. QA statement)

Test material

Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
Identification: FRET 12-0492
Chemical Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Appearance: Yellow, liquid
Storage Conditions: In the refrigerator

Test animals / tissue source

other: Reconstructed Human Corneal Epithelial Model
other: Not applicable
Details on test animals or tissues and environmental conditions:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (10 May 2017) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-¬incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (nearly 17 hours).

Test system

unchanged (no vehicle)
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
- Amount applied (volume with unit): 50 µL.

Negative control: Deionised water
Positive control: Methyl acetate
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
No data
Duration of post- treatment incubation (in vitro):
12 minute post-soak to remove any absorbed test material followed by 120 minute post-treatment incubation
Number of animals or in vitro replicates:
tested in duplicate for all test groups
Details on study design:
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm assay medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay
After post-treatment incubation of 120 minutes the MTT assay was performed.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark (17 hours) and then shaken for 2.5 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

Results and discussion

In vitro

Irritation parameter:
other: Mean relative absorbance
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 83.4% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.813 and 1.937).
• The mean relative viability of the positive control is below 50% of the negative control viability (20.6%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 1.0% and 2.3%) in the same run (for positive and negative control tissues and tissues of single test items).

This in vitro study was performed to assess the eye irritation potential of FRET 12-0492 by means of the Human Cornea Model Test.
Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured intensively, did not dye water or isopropanol, and did not prove to be a MTT reducer.
Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.
Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 20.6%, thus the validity of the test system is ensured.
Since the viability value of the test item exposed tissues did not decrease below 60%, the test item is not considered to possess an eye irritating potential.

Any other information on results incl. tables

Results after treatment for 30 minutes with FRET 12-0492 and the controls

Dose Group

Well 1
(Tissue 1/2)

Well 2 (Tissue 1/2)

Mean Absor-bance* (Tissue 1/2)

Mean Absorbance* Tissue 1 and 2

Mean Absorbance of
2 Tissues*

Rel. Absorbance [%]
Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Mean Rel. Absorbance

[% of Negative Control]**







Negative Control














Positive Control














Test Item














*         Mean of two replicate wells after blank correction

       Relative absorbance [rounded values]: (100 x (absorbance testitem/positivecontrol) / (meanabsorbancenegativecontrol)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Criteria used for interpretation of results: EU
According to the study plan followed the test item, was considered to be non-irritant.

EU CLP (1272/2008/EC)/UN GHS: Not classified for irritation.
UN GHS: Not classified for irritation.

Executive summary:

The eye irritation potential of the test substance FRET 12-0492 was assessed using the Human Cornea Model after a treatment period of 30 minutes. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013. The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test.

Relative mean tissue viability of ≤ 60 % results in a prediction of ocular irritancy. 

After a 30 minute exposure, the relative mean viability of the test item treated tissues was 83.4% and the test substance is therefore considered to be non-irritant, according to EU CLP criterial. 

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