Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July 2016 to 12 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
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:
2013
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Greenish brown crystalline powder
- Storage conditions of test material: At room temperature
- Stable until: 10 June 2021 (expiry date)

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 were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- 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.
- 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: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: None reported

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 302 to 340 mg
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects; 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 and 1 % (v/v) Foetal Bovine Serum. 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
Yes, physiological saline

POSITIVE CONTROL USED
Yes, 20 % w/v imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
302 to 340 mg for 240 ± 10 minutes

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL each of the negative and positive controls were introduced onto the epithelium of the cornea. The test material was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test material were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
- Post-exposure incubation: Yes, with sodium fluorescein

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
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 being the empirically determined illuminance through a cornea holder but with windows and medium and I being 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 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 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 5 mg Na-fluorescein/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. 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.
- Others: Possible pH effects of the test material on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

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 material induced irritation through only one of the two endpoints.

DECISION CRITERIA:
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:
In vitro score range: ≤ 3 = UN GHS No Category; > 3 but ≤ 55 = No prediction can be made; and >55 = UN GHS Category 1

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
1 812.1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
1 811.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: permeability
Run / experiment:
mean
Value:
0.041
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
The individual in vitro irritancy scores for the negative controls ranged from 1.3 to 2.4. The individual positive control in vitro irritancy scores ranged from 139 to 200. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with the test material showed opacity values ranging from 1811 to 1812 and permeability values ranging from 0.017 to 0.060. The corneas were turbid and purple coloured after the 240 minutes of treatment since the cornea absorbed the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 1812 to 1813 after 240 minutes of treatment with the test material.

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 159 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.

The test material induced serious eye damage through one endpoint (opacity), resulting in a mean in vitro irritancy score of 1812 after 240 minutes of treatment. This was probably due to the staining of the cornea by the test material, which could not be removed totally.

Any other information on results incl. tables

Table 1: Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity

Mean Permeability

Mean In vitro Irritation Score*

Negative control

1.4

0.023

1.8

Positive control

134.2

1.676

159.4

Test material

1811.5

0.041

1812.1

*Calculated using the negative control mean opacity and mean permeability values for the positive control and test material. In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Applicant's summary and conclusion

Interpretation of results:
other: Classified as Category 1 in accordance with EU criteria
Conclusions:
Under the conditions of this study, as the test material induced an IVIS >55, classification is required for serious eye damage.
Executive summary:

The hazard potential of the test material to the eye was evaluated in vitro in accordance with the standardised guideline OECD 437 under GLP conditions using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested in isolated bovine corneas through topical application for approximately 240 minutes. The test material was added neat on top of the corneas. Concurrent negative and positive controls were run using physiological saline and 20 % imidazole solution in physiological saline, respectively.

The corneas treated with the test material showed opacity values ranging from 1811 to 1812 and permeability values ranging from 0.017 to 0.060. The corneas were turbid and purple coloured after the 240 minutes of treatment since the cornea absorbed the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 1812 to 1813 after 240 minutes of treatment with the test material.

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 159 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.

The test material induced serious eye damage through one endpoint (opacity), resulting in a mean in vitro irritancy score of 1812 after 240 minutes of treatment. This was probably due to the staining of the cornea by the test material, which could not be removed totally.

Under the conditions of this study, as the test material induced an IVIS >55, classification is required for serious eye damage.