Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-02-01 to 2016-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study according to OECD guideline 437 and EU method B.47.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
(see Rationale for reliability)
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
(see Rationale for reliability)
Principles of method if other than guideline:
The study procedures were also in compliance with the following guidelines:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P, Dukic M, Alix D and Sina J F, Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): JNJ-17299412-AAA (T001599)
- Substance type: white powder
- Physical state: solid
- Analytical purity: 100.2% (base titration determination with specification >= 98%)
- Impurities: none detected
- Purity test date: no data
- Lot/batch No.: I15HB2951
- Expiration date of the lot/batch: 2017-08-09 (retest date)
- Storage condition of test material: At room temperature
- Correction factor: 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15HB2951
- Expiration date of the lot/batch: 2017-08-09
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

Test animals / tissue source

Species:
other: bovine eyes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- 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. Bovine eyes were used as soon as possible but within 4 hours after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.

- 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 (Eagle’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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: negative control (physiological saline) and positive control (20% (w/v) Imidazole solution in physiological saline)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 336.8 to 350.5 mg.
- added pure on the top of the corneas


VEHICLE
- Amount applied: 750 µl

POSITIVE CONTROL
- Amount applied: 750 µl
- Concentration: 20% (w/v) imidazole solution
Duration of treatment / exposure:
Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C
Duration of post- treatment incubation (in vitro):
After 240 ± 10 minutes of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed after the incubation period of 90 minutes ± 5 minutes following the opacity measurement.
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group.
Details on study design:
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 an air 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.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (336.8 to 350.5 mg). 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. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’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.

OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (l = 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 before/after test item treatment.
The change of 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 positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item 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 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 (Sigma-Aldrich Chemie GmbH, Germany). 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

ELECTRONIC DATA CAPTURE
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

INTERPRETATION
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.

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
73.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range 69.9 to 80.9
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
-0.4
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range -1.0 to 0.7
Irritation parameter:
other: permeability score
Run / experiment:
1
Value:
4.953
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range 4.725 to 5.343
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: -3.6 (-5.3 tot -2.3)
positive control: 156.5 (153.9 to 158.5)
test item: 73.9 (69.9 to 80.9)

mean opacity scores (range):
negative control: -3.7 (-5.4 to -2.4)
positive control: 129.1 (122.0 to 137.3)
test item: -0.4 (-1.0 to 0.7)

mean permeability scores (range):
negative control: 0.009 (0.004 to 0.017)
positive control: 1.824 (1.416 to 2.335)
test item: 4.953 (4.725 to 5.343)

The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas were slightly translucent after the 240 minutes of treatment with the test item. During rinsing a membrane of the cornea became loose. No pH effect of the test item was observed on the rinsing medium.

Interpretation:
The IVIS of all replicates was within one category.

Discussion:
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 157 (154 to 159) and within the historical positive control data range. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item induced serious eye damage through one endpoint (permeability), resulting in a mean in vitro irritancy score of 74 (70 to 81) after 240 minutes of treatment.
Since the test item induced an IVIS ≥ 55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in the report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item induced serious eye damage through one endpoint (permeability), resulting in a mean in vitro irritancy score of 74 (70 to 81) after 240 minutes of treatment. Since the test item induced an IVIS ≥ 55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in the report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.