Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase: 30 August 2018. Report Issued: 25 October 2018.
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 guideline
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 ml of either test substance, negative control, or positive control, applied to appropriate corneas.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2-hours
Number of animals or in vitro replicates:
Three replicates (corneas) per treatment (ttest substance, negative control, positive control)
Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS : Yes

NUMBER OF REPLICATES: Three per treatment

NEGATIVE CONTROL USED : Sodium chloride 0.9 % w/v

SOLVENT CONTROL USED (if applicable): No

POSITIVE CONTROL USED: Ethanol > 99.8%

APPLICATION DOSE AND EXPOSURE TIME : 10 minutes

POST-INCUBATION PERIOD: yes, two-hours.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer (calibrated on the day of the test)
- Corneal permeability: passage of sodium fluorescein dye measured using microtitre plate reader (Labtech LT-4500) at 492 nm.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As specified in the test guideline.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test Substance (mean of three replicates)
Value:
45.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS
- Visible damage: The corneas treated with the test item were partly cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.


ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

The test item was classified according to the following criteria:

 IVIS  UN GHS

 ≤ 3

 No Category

 >3; ≤ 55

No prediction can be made

 > 55

 Category 1

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The result for the test substance was within the band assigned for no prediction posssible according to the prediction model provided in the OECD 437 test guideline. Therefore no prediction of eye irritation can be made for the test substance from the results of this test.
Executive summary:

Introduction

The purpose of this test was to identify if the test item can induce serious eye damage. The method used was designed to meet the requirements of OECD Test 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 (09 October 2017).

  

Method

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

 

The test item was classified according to the prediction model as follows:

 IVIS  UN GHS

 ≤ 3

 No Category

 >3; ≤ 55

No prediction can be made

 > 55

 Category 1

Results

The in vitro irritancy scores are summarised below:

 Treatment

 In vitro Irritancy Score

 Test Item

 45.3

 Negative Control

 0.1

 Positive Control

 58.1

Conclusion

The result for the test substance was within the band assigned for no prediction posssible according to the prediction model provided in the OECD 437 test guideline. Therefore no prediction of eye irritation can be made for the test substance from the results of this test.