[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 to 26 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 437

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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 26 June 2020

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

litsea cubeba (fruit) extract – terpenes fraction

Molecular formula:

not applicable

IUPAC Name:

litsea cubeba (fruit) extract – terpenes fraction

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle obtained from the slaughterhouse (Vitelco's Hertogenbosch, The Netherlands)
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- 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 and tested the day of arrival in the laboratory.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: not specified
- Indication of any antibiotics used: not specified
- Selection and 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.
Then, 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 Duratec Analysentechnik GmbH (Hockenheim, 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.
- Quality check of the isolated corneas: 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, Duratec GmbH). 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)

Remarks:

The test material was tested neat.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL onto the epithelium of the cornea.
- Concentration (if solution): The test material was tested neat.
VEHICLE
- Amount(s) applied (volume or weight with unit): not applicable

Duration of treatment / exposure:

10±1 minutes at 32±1°C

Duration of post- treatment incubation (in vitro):

120±10 minutes at 32±1°C

Number of animals or in vitro replicates:

Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

Details on study design:

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test material 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 material over the entire cornea. Corneas were incubated in a horizontal position for 10±1 minutes at 32±1°C.

RINSING OF THE CORNEAS
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 material 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.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: 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 cornea treated with the test material 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 material 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.

- Corneal permeability: 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.
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 material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM AND INTERPRETATION OF RESULTS
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro irritancy score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
- If the test item induces an ≤ 3: UN GHS No Category
- If the test item induces an > 3; ≤ 55: No prediction can be made
- If the test item induces an > 55: UN GHS Category 1

ACCEPTANCE CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

Mean

Run / experiment:

Positive control

Value:

43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

Mean

Run / experiment:

Negative control

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

Mean

Run / experiment:

Test material

Value:

7.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

IN VITRO IRRITANCY SCORE:
- The test material induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.3 after 10 minutes of treatment. Individual IVIS values of test item-treated corneas were: 10, 5.5 and 6.5.
On the basis of these concordant results obtained on the three corneas (IVIS > 3 ≤ 55), no additional experiment was performed and no prediction on the classification can be made for eye irritation or serious eye damage.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The individual opacity scores for the negative controls ranged from 1.3 to 3.3. The individual permability scores (uncorrected) for the negative controls ranged from -0.003 to -0.001.The corneas treated with the negative control material were clear after the 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range (opacity: -2.5 to 3.70; permeability: -0.020 to 0.065) indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The individual positive control in vitro irritancy scores ranged from 34 to 56. The corneas treated with the positive control material were turbid after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control (Ethanol) was 43 and within two standard deviations of the current historical positive control mean (28-86).

It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
No pH effects of the negative and positive control were observed on the rinsing medium.

Any other information on results incl. tables

Table 7.3.2/1: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity^1	Mean Permeability^1	Mean IVIS^1, 2
Negative control	2.6	0.000	2.6
Positive control (Ethanol)	22	1.387	43
Test material	4.2	0.208	7.3

¹Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test material.

²In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made

Conclusions:

Under the experimental conditions of this study, since Litsea Cubeba oil, Terpenes fraction L62840 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP. The objective of this study was to evaluate the eye hazard potential of Litsea Cubeba oil, Terpenes fraction L62840 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).

The eye damage potential of the test material was tested through topical application for 10 minutes on isolated bovine corneas obtained from freshly slaughtered calves. The test material was applied as supplied (750 µL) directly on top of the corneas. Chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 120±10 minutes at 32±1°C. Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

 

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 43 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 material induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.3 after 10 minutes of treatment. Individual IVIS values of test item-treated corneas were: 10, 5.5 and 6.5.

 

In conclusion, under the experimental conditions of this study, since Litsea Cubeba oil, Terpenes fraction L62840 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.