Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
IUPAC Name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Aurantiol Pure
- Physical state: Yellow viscous liquid

- Expiration date of the lot/batch: February 02, 2015
Specific details on test material used for the study:
Description: Yellow viscous liquid
Batch: SC00010629
Purity/Composition: UVCB substance

Test animals / tissue source

Species:
other: Bovine eyes
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained direct from the slaughterhouse. Eyes were from young cattle slaughtered at Vitelco's Hertogenbosch, The Netherlands. The eyes were excised as soon as possible after slaughter, and used on the same day. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. Prior to use, the corneas were checked for unacceptable defects such as opacity, scratches, pigmentation and neovascularization by submersing them in physiological saline and holding them in the light. Those exhibiting defects were discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL per cornea (no corrections were made for the purity/composition of the test substance)

Duration of treatment / exposure:
10 ± 1 minutes
Observation period (in vivo):
Opacity was determined following a 120 ± 10 minute incubation period.
Number of animals or in vitro replicates:
Three corneas were used for each group.
Details on study design:
PREPARATION AND SELECTION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 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. 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. 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
The medium from the anterior compartment was removed and 750 µl of either the negative control (physiological saline) or positive control (10% (w/v) Benzalkonium Chloride) was introduced onto the epithelium of the cornea. The test substance was introduced onto the epithelium of the cornea with a cut off plastic pipette, enough to cover the epithelium completely. 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 incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium) 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.

SODIUM FLUORESCEIN APPLICATION
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 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. 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.

Results and discussion

In vitro

Results
Irritation parameter:
other: IVIS (in vitro irritancy score)
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
IVIS <3

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from -0.2 to 0.3. The individual positive control in vitro irritancy scores ranged from 130 to 138 for Benzalkonium Chloride. The corneas treated with the positive control substance were turbid after the 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range, except for one cornea. 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 133 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.

Table 1. Summary of opacity, permeability and in vitro scores

 

Treatment

Mean Opacity1

Mean Permeability1

Mean In Vitro Irritation Score1,2

Negative Control

0

0.000

0.0

Positive Control (Benzalkonium Chloride)

75

3.882

133

Aurantiol Pure

2

-0.032

1.5

1 Calculated using the negative control mean opacity and mean permeability values

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean in vitro irritation score was 1.5, therefore the test substance is considered to be non-irritating and no classification is required.
Executive summary:

Screening for the eye irritant potential of Aurantiol Pure was conducted using the Bovine Corneal Opacity and Permeability (BCOP) test (OECD Guideline 437). The test substance was applied directly to the corneas, undiluted, and incubated for 10 minutes. Negative controls (physiological saline) and positive controls (10% w/v benzalkonium chloride) were included. Opacity determinations were performed on each cornea using an opacitometer, before and after exposure. The change in opacity for each cornea was calculated. After incubation, the medium in each treatment compartment was transferred to 96-well plates and the optical density determined at 490 nm (OD490). The in vitro irritancy score (IVIS) for each group were calculated by adding the mean opacity value to (15 x mean OD490 value). The negative control (physiological saline) 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, except the response of one cornea. This resulted in a mean permeability value just above the upper limit of the range. However since the other two values were less than the upper limit of the laboratory historical range, this deviation in the permeability score had no effect on the results of the study. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 133 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.

Aurantiol Pure did not cause ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment. It is concluded that, under the experimental conditions, Aurantiol Pure is a non-irritant in the BCOP test. Since the IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.