Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-acetyl-3-(4-hexadecoxyphenyl)-1,4-benzoquinone
EC Number:
947-565-8
Molecular formula:
C30 H42 O4
IUPAC Name:
2-acetyl-3-(4-hexadecoxyphenyl)-1,4-benzoquinone

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- 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.
- Number of animals:
/
- Characteristics of donor animals (e.g. age, sex, weight):
Young cattle.
- 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) Fetal 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.
- Time interval prior to initiating testing:
not specified
- indication of any existing defects or lesions in ocular tissue samples:
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.
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.
- Indication of any antibiotics used:
/

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µl of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) suspension of the test item was introduced onto the epithelium of the cornea.
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 240 +- 10 minutes at 32 +- 1°C.
Observation period (in vivo):
not relevant
Duration of post- treatment incubation (in vitro):
not relevant
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group.
Details on study design:
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.
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) Fetal 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.
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, 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.
NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.
NEGATIVE CONTROL USED
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
SOLVENT CONTROL USED (if applicable)
/
POSITIVE CONTROL USED
The positive control was a 20% (w/v) Imidazole (Merck KGaA, Darmstadt, Germany) solution prepared in physiological saline.
APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 l of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) suspension of the test item was introduced onto the epithelium of the cornea.
TREATMENT METHOD: not specified

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
- POST-EXPOSURE INCUBATION: not relevant

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 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.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
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 l 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.
- Others (e.g, pertinent visual observations, histopathology): (please specify)
none

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro 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 item induced irritation through only one of the two endpoints.
DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
IVIS ≤3: No Category
IVIS > 3; ≤ 55: No prediction can be made
IVIS >55: Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
-1.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
0.037
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
-0.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The corneas treated with V134434 showed opacity values ranging from -1.6 to -0.2 and permeability values ranging from 0.024 to 0.052. The corneas were translucent after the 240 minutes of treatment with V134434. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.1 to 0.3 after 240 minutes of treatment with V134434.

The individual in vitro irritancy scores for the negative controls ranged from 2.8 to 3.2. The corneas treated with the negative control item were translucent after the 240 minutes of treatment. All values were within the historical control database. The individual positive control in vitro irritancy scores ranged from 159 to 194. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, since V134434 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 V134434 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 V134434 was tested through topical application for approximately 240 minutes.

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

Batch v134434/CA of V134434 was an orange-red powder. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure 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 (20% (w/v) Imidazole) was 177 and 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.

V134434 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.5 after 4 hours of treatment.

In conclusion, since V134434induced an IVIS ≤ 3, no classification is required for eyeirritation or serious eye damage.