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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
other: colourless limpid liquid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
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.

Test system

unchanged (no vehicle)
other: yes, physiological saline
Amount / concentration applied:
Duration of treatment / exposure:
10 min
Details on study design:
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 (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.

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 either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance 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 substance over the entire cornea.
Corneas were incubated in a horizontal position for 10 ¿ 1 minutes at 32+/- 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM. Possible pH effects of the test substance 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 the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

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, Darmstadt, 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/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 Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

In vitro irritancy score
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 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 UN GHS
= 3 No Category
> 3; = 55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Vehicle controls validity:
not examined
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
LUPEROX V10 was tested neat.
The individual in vitro irritancy scores for the negative controls ranged from -3.0 to 1.0. The individual positive control in vitro irritancy scores ranged from 131 to 153 for Benzalkonium Chloride. The corneas treated with the positive control substance were turbid after the 10 minutes of treatment.
The corneas treated with LUPEROX V10 showed opacity values ranging from 1 to 3 and permeability values ranging from -0.003 to 0.005. The corneas were clear after the 10 minutes of treatment with LUPEROX V10. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 1.0 to 3.1 after 10 minutes of treatment with LUPEROX V10.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
LUPEROX V10 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 10 minutes of treatment.Since LUPEROX V10 induced an IVIS <= 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

The eye irritancy potential of LUPEROX V10 was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test) performed following the OECD guideline No. 437. LUPEROX V10 was tested through topical application for 10 ± 1 minutes. LUPEROX V10 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. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 144 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. LUPEROX V10 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 10 minutes of treatment. LUPEROX V10 is not irritant in the Bovine Corneal Opacity and Permeability test.