Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-15 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
Adopted July 26, 2013
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
white to brownish
Details on test material:
Batch 151222

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.
- Number of animals:/
- Characteristics of donor animals (e.g. age, sex, weight):/
- 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:
- 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.
- Indication of any antibiotics used: /

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
Since no workable suspension of CH02906 in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.
Amount / concentration applied:
CH02906 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (individual values 363.7, 324.2 and
363.7 mg).
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 240 +- 10 minutes at 32 +- 1°C.
Number of animals or in vitro replicates:
3
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) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder, closed chamber method) 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
20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.
APPLICATION DOSE AND EXPOSURE TIME
CH02906 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (individual values 363.7, 324.2 and
363.7 mg). Corneas were incubated in a horizontal position for 240  10 minutes at 32  1C.
TREATMENT METHOD: The isolated corneas were mounted in a corneal holder (one cornea per holder, closed chamber method) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 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 (Earle’s Minimum Essential Medium Life Technologies).
- POST-EXPOSURE INCUBATION: /

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.
- Corneal permeability: 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, serial number 1004004186). 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.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: in vitro score range: <= 3: no category, >3;<=55: no prediction possible, >55: category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
Mean of three replicates
Value:
ca. 115.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Mean permeability
Run / experiment:
Mean of three replicates
Value:
ca. 1.813
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three replicates
Value:
ca. 142.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The corneas treated with CH02906 showed opacity values ranging from 50 to 235 and permeability values ranging from 0.873 to 3.577. The corneas were turbid with spots after the 240 minutes of treatment with CH02906. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 76 to 250 after 240 minutes of treatment with CH02906. The test item could not completely be removed from the third cornea showing an IVIS of 250. Since all three corneas had an IVIS≥ 55, this had no influence on the outcome of the test.

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 149 and within two standard deviations of the current historical positive control mean (APPENDIX 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
CH02906 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 143 after 240 minutes of treatment.
Since CH02906 induced an IVIS ≥ 55, it is concluded that CH02906 induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

Evaluation of the eye hazard potential of CH02906 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 CH02906 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 151222 of CH02906 was a white to brownish powder with a purity of 99.9%. 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 meanin vitroirritancy score of the positive control (20% (w/v) Imidazole) was 149 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.

CH02906 induced serious eye damage through both endpoints, resulting in a meanin vitroirritancy score of 143 after 240 minutes of treatment.

Since CH02906induced an IVIS ≥ 55, it is concluded thatCH02906 induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.