Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Appearance: solid, crystalline, colourless powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Characteristics of donor animals (e.g. age, sex, weight): at least 9 months old donor cattle
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: The corneae were directly used in the BCOP test on the same day.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularisation, pigmentation, opacity and scratches were discarded.

Test system

Vehicle:
other: Saline (0.9% NaCl in deionised water)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20 % w/v

VEHICLE
- Amount(s) applied: 0.75 mL
- Concentration (if solution): 0.9 %
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1°C for permeability determination.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
- All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularisation, pigmentation, opacity and scratches were discarded. 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. The corneae were directly used in the BCOP test on the same day.
- Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. 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 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. Care was taken to assure that no air bubbles were present within the compartments.
- For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1°C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test material and for the negative and positive controls, respectively.

NUMBER OF REPLICATES: 3

VEHICLE CONTROL USED: 0.9% NaCl (saline)

POSITIVE CONTROL USED: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of 20% test material suspension in saline for 240 minutes.

TREATMENT METHOD:
- The anterior compartment received the test material suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1°C in the water-bath. The incubation time lasted 240 minutes.

REMOVAL OF TEST MATERIAL
- The test material or the control materials, respectively, were each rinsed off from the according application sides with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240) using an opacitometer [OP_KiT opacitometer (Electro Design, 63-Riom, France)].

POST-EXPOSURE INCUBATION:
- Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1°C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate.
- The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
- Opacity: The change of the opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: The corrected OD490 value of each cornea treated with positive control or test material is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
- IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test material:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test material is classified into the following Category according to OECD guideline 437:
IVIS ≤ 3: No category
IVIS > 3 to ≤ 55: No prediction can be made
IVIS > 55: Category 1

VALIDITY OF THE TEST
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
1.09
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
- For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.18).
- The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.30) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
- The test material was tested as suspension. Relative to the negative control, the test material did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.09 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test material is not categorised.

Any other information on results incl. tables

Table 1: Summary of results after 240 minutes treatment.

Test group

Mean IVIS

Proposed Classification

Negative control

1.18

Not categorised

Positive control

98.30

Category 1

Test material

1.09

Not categorised

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

The potential of the test material to cause eye irritation or damage was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions, with a BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test material, the positive and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1°C. After the incubation phase, the test material as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1°C.

For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.18). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.30) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test material did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.09 (threshold for serious eye damage: IVIS > 55).

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.