Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jan 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test method is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it.The permeability, as a result of the irritation potential of the test item, is determined using sodium fluorescein solution. The comparison of the opacity before and after the exposure to the test item and the determination of the permeability after the treatment provide an indication of the damaging effect of the test item. For this purpose the induction of opacity and increased permeability in an isolated bovine cornea after application of the test item was measured. The results of both criteria were combined. The resulting in vitro irritation factor (IVIS) was compared with the classification according to the UN GHS System.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Schlachthof Bensheim, Bensheim, Germany
- Donor animals: at least 9 month old
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes. The cornea were directly used in the BCOP on the same day.
- Transport medium and temperature conditions: Hank's Buffered Salt Solution (HBSS) supplemented with penicillin/streptomycin at ambient temperature.

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.
- Description of the cornea holder: The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively.
- Test medium used in the cornea holder: The incubation medium consisted of MEM, supplemented with 1.1 g/500 mL sodium bicarbonate, 5 mL/500 mL L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin. Immediately before starting the test, MEM was supplemented with 1% fetal calf serum.
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity >7 was discarded.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: OP_KiT opacitometer, Electro Design, France

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL

POSITIVE SUBSTANCE
- Substance: 2-ethoxyethanol
- Amount(s) applied in the test: 0.75 mL

NEGATIVE CONTROL
- 0.9% NaCl (w/v) solution in deionised water (saline)
Duration of treatment / exposure:
10 min at 32±1 °C
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
3
Details on study design:
TEST CONDITIONS
- Short description of the method used: The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. The anterior compartment received the test item or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test item was rinsed off from the application side with saline.

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: After the test item was rinsed off from the application side, the corneae were incubated for further 2 hours in a vertical position, followed by a second opacity reading (t130).
- Specification of the device: OP_KiT opacitometer, Electro Design, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by sodium fluorescein solution. Corneae were incubated again in horizontal position for 90 ± 10 min in a water-bath at 32 ± 1 °C. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with a spectrophotometer as optical density at 490 nm (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (0.4% (w/v); dissolved in HBSS)
- Incubation time: 90 min at 32 ± 1 °C
- Treatment for measuring: Complete medium from the posterior compartment was removed, well mixed, transferred into a 96 well plate and OD490 was determined.
- Specification of the spectrophotometer: Versamax Molecular Devices

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean out of 3
Value:
-0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Run / experiment:
number 1
Value:
0.009
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Run / experiment:
number 2
Value:
0.004
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Run / experiment:
number 3
Value:
0.004
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
mean out of 3
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
- Acceptance criteria met for positive control: yes; The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging.

Any other information on results incl. tables

Table 1. Historical Data

 

Positive control

Negative control

Mean IVIS

71.85

1.07

SD

17.57

0.71

Range of IVIS

33.59 – 153.20

0.00 – 2.84

Values of 274 studies with liquid test items performed from February 2007 until December 2014.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not classified