2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2019 - 13 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted October 09, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

EC Number:

278-051-5

EC Name:

2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Cas Number:

75005-95-7

Molecular formula:

C28H30O3

IUPAC Name:

2-ethylhexyl 2-{[1,1'-biphenyl]-4-carbonyl}benzoate

Test material form:

liquid

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
CAS number: 75005-95-7

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Preparation of Corneas:The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Cornea Selection and Opacity Reading: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item (excessive amount) was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

Duration of post- treatment incubation (in vitro):

Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

3

Details on study design:

TEST ITEM PREPARATION
No correction was made for the purity/composition of the test item.
The test item was tested neat.


TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item (excessive amount) was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a
horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.


OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:

Opacity = (( I0 / I ) - 0.9894) / 0.0251.

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was
calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.


APPLICATION OF SODIUM FLUORESCEIN
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.


PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

Mean Opacity

Run / experiment:

Test item

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Mean Permeability

Run / experiment:

Test item

Value:

0.025

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

Mean In vitro Irritation Score

Run / experiment:

Test item

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

cornea opacity score

Remarks:

Mean Opacity

Run / experiment:

Negative control

Value:

2.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

other: Mean Permeability

Run / experiment:

Negative control

Value:

0.003

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

in vitro irritation score

Remarks:

Mean In vitro Irritation Score

Run / experiment:

Negative control

Value:

2.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

cornea opacity score

Remarks:

Mean Opacity

Run / experiment:

Positive control (Ethanol)

Value:

20

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation parameter:

other: Mean Permeability

Run / experiment:

Positive control (Ethanol)

Value:

1.888

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 9

Irritation parameter:

in vitro irritation score

Remarks:

Mean In vitro Irritation Score

Run / experiment:

Positive control (Ethanol)

Value:

48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Results calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Any other information on results incl. tables

The test item was tested neat.  

The above in-vitro data summarizes the opacity, permeability and in vitro irritancy scores of the test item and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 - 5 in the attached appendix.  

The individual in vitro irritancy scores for the negative controls ranged from 2.4 to 2.9. The corneas treated with the negative control item were clear after the 10 minutes of treatment.

The individual positive control in vitro irritancy scores ranged from 40 to 54 (Appendix, Table 5).  The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from -0.8 to 4.0 and permeability values ranging from 0.009 to 0.057. The corneas were translucent/clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.  Hence, the in vitro irritancy scores ranged from 0.0 to 4.1 after 10 minutes of treatment with the test item.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.  The mean in vitro irritancy score of the positive control (Ethanol) was 48 and within two standard deviations of the current historical positive control mean (Appendix, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 10 minutes of treatment. Although one of the test item treated corneas resulted in an IVIS score of 4.1, the IVIS score was only slightly above the cut-off value of 3 and was close to the mean IVIS score of 1.7.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

The test item was a colourless to pale yellow liquid with a purity of 96.27%. The test item was applied as it is (excessive amount) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 10 minutes of treatment.

In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.