Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 28, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 437, EU Method B.47 and other internationally accepted guidelines, in compliance with GLP.

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
Qualifier:
according to
Guideline:
other: Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006 (ICCVAM).
Deviations:
no
Qualifier:
according to
Guideline:
other: NVITTOX) study plan 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
Deviations:
no
Qualifier:
according to
Guideline:
other: Gautheron P., Dukic M., Alix D. and Sina J.F. (1992). Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
Paste
Details on test material:
- Name of test material (as cited in study report): Vegetable or animal oil, hydrogenated, reaction products with diethanolamine, maleic anhydride and sodium sulphite
- Lot No.: CH 192435/01
- Purity: Not applicable (UVCB)
- Organic Carbon: 30.2%
- Hydrotox No.: 15/5241
- Origin: DACC
- Stability: Stable within the durability and with appropriate storage conditions
- Solubility in water: ~1 g/L
- Appearance: Light beige liquid/paste
- Storage conditions: Ambient temperature, dark, dry
- Expiration date: July 15, 2015
- Safety directions: H315, H319. Use protective clothing: gloves and protection goggles.

Test animals / tissue source

Species:
other: Bovine eyes
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- 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.

Test system

Vehicle:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: Due to the consistency of the test substance, an excess amount was applied
Duration of treatment / exposure:
10 min
Details on study design:
Negative control: 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.

Positive control: 10% (w/v) Benzalkonium Chloride (Merck KGaA, Darmstadt, Germany) [CAS Number 63449-41-2] solution prepared in physiological saline.

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 (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 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 h 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 an excessive amount of the 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 mins 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, Life Technologies) 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 mins 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 (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 mins 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.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
10 minutes
Value:
ca. -1.8
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1 Summary of opacity, permeability and in vitro scores

Treatment

Mean

opacity1

Mean

permeability1

Mean in vitro irritation score1, 2

Negative control

-0.3

0.000

-0.3

Positive control

(Benzalkonium Chloride)

103.0

2.481

140.2

Test substance

-2.0

0.011

-1.8

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:
other: CLP criteria not met
Conclusions:
The test substance is not-irritating to the eyes.
Executive summary:

The eye hazard potential of the test substance was evaluated in a bovine corneal opacity test conducted according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The test consisted of the topical application of undiluted test substance on the epithelium of a bovine cornea for 10 min. The test was carried out using three replicates each for test substance, negative control and positive control. After exposure, the cornea was thoroughly rinsed and incubated for 2 h with fresh medium, followed by opacity measurement. The permeability of the cornea was then determined after a 90 min incubation period with sodium fluorescein. The test substance did not induce ocular irritation in both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score (IVIS) of -1.8 after 10 min of treatment. 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 (10% (w/v) benzalkonium chloride) was 140, 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. Hence, based on the results of the study, the test substance was considered to be non-irritating to eyes (Eurlings IMJ, 2015b).