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Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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)
Version / remarks:
July, 2013
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Test item: Leuco Sulfur Blue 13
Appearance: Bluish black, solid
CAS No: 12262-26-9

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: 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.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: Yes

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
The test item was tested as a 20 % suspension (w/v) in saline using sonication for 10 minutes. Prior to treatment of the corneae the pH-value of the test item solution or suspension was determined (pH 7.65).

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: The test item was tested as a 20 % suspension (w/v) in saline using sonication for 10 minutes.

VEHICLE
- Amount applied: 0.75 mL
- Concentration: Saline (0.9 % NaCl in deionised water)
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, 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 OEDC guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and 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 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).

QUALITY CHECK OF THE ISOLATED CORNEAS
The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

NUMBER OF REPLICATES
3 corneae per group (test item, negative control, positive control)

SOLVENT CONTROL USED
Saline (0.9 % NaCl in deionised water)

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

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL, 240 min

TREATMENT METHOD:
closed chamber

POST-INCUBATION PERIOD:
no

REMOVAL OF TEST SUBSTANCE
the test item or the control items, respectively, were each rinsed off from the according application sides with saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: via opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France))
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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 item:
IVIS = (opacity value – 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 score obtained, the test item is classified into the following category according to OECD guideline 437:

DECISION CRITERIA
IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean
Run / experiment:
1-3
Value:
1.7
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Table 1: Results after 240 Minutes Treatment Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

Standard Deviation of in vitro Score

 

 

Mean

 

Mean

 

 

 

 

Negative Control

0

0.00

0.054

0.057

0.81

0.86

Not categorized

0.04

0

0.059

0.89

0

0.058

0.87

Positive Control

121.00*

0.019*

121.29

119.53

Category 1

2.91

116.00*

0.011*

116.17

121.00*

0.009*

121.14

Test Item

2.00*

-0.004*

1.94

1.70

Not categorized

0.57

1.00*

0.003*

1.05

2.00*

0.007*

2.11

*corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro eye irritation assay (BCOP) according to OECD 437, the test item did not show eye irritating properties.
Executive summary:

The eye irritating properties of the test item were assessed in a BCOP assay using fresh bovine corneae, according to OECD guideline 437.

After an initial opacity measurement of the fresh bovine corneae (t0), the 20 % (w/v) suspension in saline of the test item, 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 item as well as the positive and the negative controls were each rinsed off from the corneae and opacity was measured again (t240). After the opacity measurements, permeability of the corneae was determined by measuring the transfer of sodium fluorescein spectrophotometrically after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

For the negative control (saline, 0.9 % NaCl) neither an increase of opacity nor permeability of the corneae was observed (mean IVIS =0.86). The positive control (10 % (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =119.53) corresponding to a classification as serious eye damaging (UNGHS Category 1). All acceptability criteria were fulfilled.

The test item was tested as suspension. Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity. The calculated mean IVIS was 1.70 (threshold for serious eye damage: IVIS > 55). According to the OECD guideline 437, the test item is not categorized (UN GHS No Category).