Methyl 4-[2-[4-(5-methyl-2-benzoxazolyl)phenyl]vinyl]benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April from 18th to 19th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fluorescent Brightener 371

IUPAC Name:

Fluorescent Brightener 371

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- System used: the EpiOcular™ human cell construct (MatTek Corporation), three-dimensional human cornea model. The model is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells.
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
- Storage: the EpiOcular™ (OCL-200-EIT) units were stored in refrigerator (2-8 °C) until the preparation of tissues for treatment is started.

EpiOcular KIT - SUITABILITY
- The air bubbles between the agarose gel and insert: There were no air bubbles observed under the inserts.
- Cooler boxes: the condition of the cooler boxes was checked, because the frozen cooler box can guarantee that the temperature of kit was between 2-8°C during the transport.
The kit was found to be in good order at reception.

PREPARATION OF TISSUES
- Arrival: after the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes.
- Preparation of first pre-incubation: the Assay Medium was pre-warmed to 37 °C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 ml per well).
- First pre-incubation: the insert was transferred aseptically into the 6-well plates and pre-incubated at 37 ± 2 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere for one hour in the Assay Medium.
- First pre-incubation duration: one hour
- Pre-incubation: 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 (16 - 24 hours).
- Pre-treatment: after the overnight incubation, the tissues were pre-wetted with approximately 20 μl of Ca2+ Mg2 + Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied in its original form (50 mg on top of 0.6 cm2 tissue; approx. 83.3 mg/cm2), no formulation was required.

Duration of treatment / exposure:

6 hours (± 15 min)

Duration of post- treatment incubation (in vitro):

Post-Soak incubation: 25 ± 2 minute.
Post-treatment incubation: 18 hours ± 15 minutes.

Number of animals or in vitro replicates:

2 replicates per test item, 2 replicates negative controls, 2 replicates positive controls and 2 replicates colour controls

Details on study design:

TEST PROCEDURES
Exposure conditions: standard culture conditions, at 37 ± 2 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere.

Rinsing: after the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca2+ Mg2+ Free-DPBS. Three clean beakers (glass or plastic with minimal 150 ml capacity), containing a minimum of 100 ml each of Ca2+ Mg2+ Free-DPBS were used per test item. 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 (paper towel, gauze, etc.) and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was 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 tissue was 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. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

Post-Soak and Post-incubation: after rinsing, the tissues were transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 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 insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 ml of warm (37 °C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

MTT Test After Post-incubation
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µl of 1 mg/ml MTT per well) and then incubated for 3 hours (± 15 min) at 37 ± 2 °C in an incubator with 5 ± 1 % CO2 protected from light, ≥ 95 % humidified atmosphere.

Formazan Extraction
Inserts were removed from the 24-well plate after 3 hours ± 15 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm (between the plate cover and upper edge of the wells).
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (120 rpm) for approximately 3 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.

Cell Viability Measurements
Following the formazan extraction, 200 µl sample(s) from each tube (preferably 2×200 µl if possible) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm (± 10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using isopropanol solutions as the blank (8×200 µl).

CONTROLS
Positive and Negative Control: a volume of 50 µl positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette.
- Additional controls for dyes and chemicals able to colour the tissue: two additional test item treated tissues were used for the non-specific OD evaluation.

CHECK OF TEST SYSTEM SUITABILITY
Indicator for Potential False Viability
Optical properties of the test item or its chemical action on MTT, isopropanol and water were assayed for possible interfere with the assay.

Check-method for Possible Direct MTT Reduction by the Test Item
Approximately 50 mg test item was added to 1 ml MTT 1 mg/ml solution in a tube and the mixture was incubated in the dark at 37 ± 2 °C, 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The mixture was incubated for approximately three hours and then any colour change observed.

Check-method to Detect the Colouring Potential of Test Item
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. For this purpose approximately 50 mg test item was added to 1.0 ml of water in a tube and the mixture was incubated in the dark at 37 ± 2 °C, 5 % ± 1 CO2, ≥ 95 % humidified atmosphere for about one hour and the colour was checked afterwards (unaided eye assessment).
Furthermore, approximately 50 mg test article was added to 2 ml isopropanol, the same amount as used for MTT extraction, and was incubated in tube. The tube was placed on an orbital plate shaker and shaken (120 rpm) for 2 to 3 hours at room temperature and then colour checked (unaided eye assessment).
The test item was tested for its ability to absorb significantly light at the wavelength (570 nm) used for the MTT determination. The used solution was same which was prepared during the check test of examination of possible color change impact of isopropanol. According to the results of the OD measurement the use of additional controls was necessary. Two additional test item treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps than other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD readings were made following the same conditions as for other tissues.

ASSAY ACCEPTANCE CRITERIA
- The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
- The acceptable percentage viability for positive control (mean of two tissues) is 6 hours exposure: below 50 % of control viability (solids).
- The difference of viability between the two relating tissues of a single chemical is < 20 % in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

INTERPRETATION OF RESULTS
Mean tissue viability % is ≤ 60 %: test item able to cause eye irritation
Mean tissue viability % is > 60 %: test item not irritating

Results and discussion

In vitro

Results

Irritation parameter:

other: viability (%)

Run / experiment:

mean of replicates

Value:

71

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 71 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to eye.

CHECK OF TEST SYSTEM SUITABILITY
No colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item. The test item did not interact with MTT, therefore additional controls and data corrections were not necessary. A false estimation of viability can be precluded.

Colouring potential of test item
The test item showed no ability to become coloured in contact with water. However, it showed highly opacity solution after contact with isopropanol. So photometric assessment at 570 nm was conducted (it was done in isopropanol). Mean OD (measured at 570 nm) of the test item was determined as 1.0038 (100.38 %). This is above 5 %, thus, additional controls were necessary:
Mean OD (measured at 570 nm) of these tissues was determined as 0.014 at 6 hours exposure. The Non Specific Colour % (NSCliving %) was calculated as 0.84 % at 6 hours exposure. Therefore additional data calculation was not necessary. A false estimation of the viability can be precluded in all occasions.

CONTROLS
Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

VALIDITY CRITERIA
- The mean OD value of the two negative control tissues was: 1.680
- The positive control result showed 8 % viability at 6 hours exposure.
- The difference of viability between the two tissue replicates 0.1 % to 17.9 %
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Any other information on results incl. tables

OD values and viability percentages of the test item

Optical Density (OD)		Viability (%)	Δ%
1	1.043	62	17.9
2	1.345	80
mean	1.194	71	-
standard deviation (SD)		12.68	-

OD values and NSC percentage of additional control

Optical Density (OD)		Non Specific Colour % (NSCliving %)	Δ %
1	0.015	0.84	0.1
2	0.013
mean	0.014		-

Remark: Δ % The difference of viability between the two relating tissues

OD values and viability percentages of the controls

Controls	Optical Density (OD)		Viability (%)	Δ %
Negative Control Sterile deionized water	1	1.720	102	4.7
	2	1.640	98
	mean	1.680	100	-
Positive Control Methyl acetate	1	0.112	7	3.3
	2	0.168	10
	mean	0.140	8	-

Applicant's summary and conclusion

Interpretation of results:

other: not classified. according to the CLP Regulation (EC) 1272/2008

Conclusions:

Not eye irritating

Executive summary:

The purpose of the study was to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to the OECD testing guideline 492.

Before treatment the tissues were pre-wetted with approximately 20 μl of Ca2+ Mg2+ Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca2+ Mg2+ Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37 ± 2 °C in an incubator with 5 ± 1 % CO2 protected from light, ≥ 95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item showed no ability to become coloured in contact with water. However, it showed highly opacity solution after contact with isopropanol, so it was tested for its ability to absorb significantly light at the wavelength (570 nm) used for the MTT determination. According to the results of the OD measurement the use of additional color controls was necessary.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 71 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. Thus, the test item is considered as non-irritant to eye.

Conclusion

Not eye irritating