Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 January 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:
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)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance / physical state: Clear, colourless, liquid
- Storage conditions: Room temperature

Test animals / tissue source

Species:
cattle

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 μL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 ± 10 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
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.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

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) 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.

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 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.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
- The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (Ethanol) or test item 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 item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C.
- After 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 item 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 completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

OPACITY MEASUREMENT
- 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 using the equation Opacity = [(I0/I) – 0.9894] / 0.0251 where I0 = the empirically determined illuminance through a cornea holder but with windows and medium; 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 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.

APPLICATION OF SODIUM FLUORESCEIN
- 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.

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 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.

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

INTERPRETATION
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score using the equation In vitro irritancy score (IVIS) = mean opacity value + (15 * 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.

COMPUTERISED SYSTEMS
- Optical Density Measurement: Magellan Tracker version 7.0
- Temperature (laboratory facilities) data collection: REES Centron version SQL 2.0

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean
Value:
1.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS
- Table 1 (attached) summarises the opacity, permeability and in vitro irritancy scores of the test item and the controls.
- The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 to 5 (attached).
- The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.1.
- The individual positive control in vitro irritancy scores ranged from 48 to 88 (see Table 5, attached). The corneas treated with the positive control item were turbid after the 10 minutes
of treatment.
- The corneas treated with test item showed opacity values ranging from 0.6 to 1.7 and permeability values ranging from 0.015 to 0.032. The corneas were clear after the 10 minutes
of treatment.
- No pH effect of the test item was observed on the rinsing medium.
- The in vitro irritancy scores ranged from 1.1 to 2.1 resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment with the test material.

Any other information on results incl. tables

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 (Ethanol) was 64 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.

- The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since the test iteminduced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

GUIDELINE

The objective of this study was to evaluate the eye hazard potential of the test item 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 investigation was conducted in accordance with 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, (adopted July 26, 2013).

 

METHODS

This report describes the potency of chemicals to induce serious eye damage using isolated

bovine corneas. Eye damage was tested through topical application for 10 minutes. The test item was a clear colourless liquid. The test item was applied as it is (750 μL) directly 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.

 

RESULTS

The mean in vitro irritancy score of the positive control (Ethanol) was 64 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 item did not induce ocular irritation through both endpoints, resulting in a mean in vitro

irritancy score of 1.4 after 10 minutes of treatment.

 

CONCLUSION

Since the test iteminduced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.