Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD method without significant deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Identification: D-C16
Appearance: White crystal
Batch: 14-002
Purity/Composition: 99.3%
Test substance storage: At room temperature, dark place, airtight, Stable under storage conditions until 24 October 2019 (expiry date)
Purity/composition correction factor: No correction factor required
Test substance handling: No specific handling conditions required
pH (1% in water, indicative range): 5.6 – 5.3 (determined by WIL Research Europe B.V. )

Test animals / tissue source

Species:
other: bovine eyes from slaughter house
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
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, Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) 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 controls (physiological saline) and positive controls (20% (w/v) imidazole) were used.
Amount / concentration applied:
No correction was made for the purity/composition of the test substance. Since no workable suspension of D-C16 in physiological saline could be obtained, the test substance was used as delivered by the sponsor and added pure on top of the corneas.
Duration of treatment / exposure:
not applicable, incubation of corneas was performed for 240 ±10 minutes at 32 ±1 °C.
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
3 corneas per group were used
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 (OP-KIT, MC2, Clermont-Ferrand, France). 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. D-C16 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (321.7 to 358.4 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, Invitrogen Corporation). Possible pH effects of the test substance 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 opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. 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 positive control or test substance 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 substance 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 (Merck) 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.
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 substance induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range ≤ 3  No UN GHS Category
In vitro score range > 3; ≤ 55  No UN GHS prediction can be made
In vitro score range >55  UN GHS Category 1
Acceptability of the assay:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean value.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
6.12. List of deviations
In the solvent control measurement, one cornea was not placed correctly in the holder and this measurement was excluded from the IVIS determination. Since the other two measurements were within the historical data, this deviation in the IVIS determination had no influence on the study results. The study integrity was not adversely affected by the deviation.
Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.

Results and discussion

In vivo

Results
Irritation parameter:
other: in vitro irritation score (IVIS)
Basis:
mean
Score:
-0.5
Max. score:
135.4
Reversibility:
other: not applicable
Remarks on result:
other: The max. score is taken from positive control results; negative control was -0.5.
Irritant / corrosive response data:
D-C16 was tested as it is.
The table “Summary of opacity, permeability and in vitro scores “summarizes the opacity, permeability and in vitro irritancy scores of D-C16 and the controls.
The individual in vitro irritancy scores for the negative controls ranged from -1.1 to 0.1. In the solvent control measurement, one cornea was not placed correctly in the holder and was excluded from the IVIS determination. Since the other two measurements were within the historical control data base, this deviation in the IVIS score determination had no influence on the study results. The individual positive control in vitro irritancy scores ranged from 130 to 141. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with D-C16 showed opacity values ranging from -1.0 to 0.0 and permeability values ranging from 0.000 to 0.028. The corneas were clear after the 240 minutes of treatment with D-C16. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.0 to 0.4 after 240 minutes of treatment with D-C16.
Other effects:
No effect on pH was observed

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity

Mean Permeability

Mean In vitro Irritation Score*

Negative control

-0.5

0.000

-0.5

Positive control

107.0

1.896

135.4

D-C16

-0.7

0.013

-0.5

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

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
D-C16 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.5 after 240 minutes of treatment.
Since D-C16 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

Evaluation of the eye hazard potential of D-C16 was performed using the Bovine Corneal Opacity and Permeability test (BCOP test, OECD 429). The report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of D-C16 was tested through topical application for approximately 240 minutes. 

Batch 14-002 of D-C16 was a white crystal with a purity of 99.3%. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (321.7 to 358.4 mg).

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 135 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

D-C16 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.5 after 240 minutes of treatment. Since D-C16 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.