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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:
key study
Study period:
2.9.2010 - 30.9.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Although the study was not performed under GLP principles, it was carried out according to generally valid and internationally accepted testing guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Test Guideline No. 437, Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Adopted 7th September 2009.
Principles of method if other than guideline:
The Bovine Corneal Opacity and Permeability test method (BCOP) is an in vitro test method that can be used to classify substances as 'ocular corrosives and severe irritants'. The BCOP uses isolated corneas from the eyes of cattle slaughtered for commercial purposes, thus avoiding the use of laboratory animals. Each treatment group (test substance, negative/positive controls) consists of a minimum of three eyes where the cornea has been excised and mounted to a holder. Depending on the physical nature and chemical characteristics of the test substance, different methods can be used for its application since the critical factor is ensuring that the test substance adequately covers the epithelial surface. Effects to the cornea are measured as opacity and permeability, which when combined gives an In Vitro Irritancy Score (IVIS) for each treatment group. A substance that induces an IVIS superior or equal to 55.1 is defined as a corrosive or severe irritant.

GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tar, brown-coal, low-temp.
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Smiles notation: not known - UVCB substance
- InChl: not known - UVCB substance
- Substance type: technical product
- Physical state: viscous liquid
- Lot/batch No.: T201E
- Expiration date of the lot/batch: 31.3.2011
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark


Test system

Vehicle:
unchanged (no vehicle)
Details on study design:
Procedure scheme:
Selection of corneas, mounting in holders → incubation with EMEM (Eagle`s Minimum Essential Medium 1hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity → treatment by positive and negative control substance and test substance (incubation 10 min.) → washing epithelium, incubation 2 hour (32 ± 1°C), measurement of opacity after application → application of sodium fluorescein (4 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of absorbance (490 nm).

Treatment groups:
The test was performed using nine isolated bovine corneas. Three corneas for each group – positive control group, negative control group and test substance group.

Control substances
Concurrent negative controls and positive controls were included in experiment. The control group was included in the BCOP test method so that nonspecific changes in the test system could be detected and to provide a baseline for the assay endpoints
Negative control substance: 0.9% sodium chloride solution
Positive control substance: 1% NaOH solution

Aplication of the substance:
Open-chamber method was used, because the test substance was viscous liquid. The test substance (enough test substance to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the micropipet. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.

Endpoints Measured
Opacity - the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale.
Permeability - the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using UV/VIS visible light spectrophotometry (at 490 nm).

Evaluation of results
Once the opacity and mean permeability (A490 ) values had been corrected for background opacity and the negative control permeability A490 values, the mean opacity and permeability A490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:

IVIS = mean opacity value + (15 x mean permeability A490 value)

Results and discussion

In vivo

Results
Irritation parameter:
other: IVIS (In Vitro Irritancy Score ) - see section Details on study design
Basis:
other: see section Details on study design
Time point:
other: 10 min.
Score:
27.03
Reversibility:
not specified

Any other information on results incl. tables

Opacity values

 

Application

substance

Cornea No.

Baseline opacity

Opacity after treatment

Opacity difference

Mean

Negative control

(0.9% sodium chloride solution)

1

7

8

1

0.33

4

7

7

0

12

6

6

0

Positive control

(1%NaOH solution)

2

7

118

111

107.00

 

7

5

104

99

11

7

118

111

Test substance

(Tar, brown-coal, low-temp.)

5

7

28

21

22.67

10

5

26

21

15

7

33

26

 

Absorbance values

 

Application

substance

Cornea No.

Absorbance

(490 nm)

Mean

Negative control

(0.9% sodium chloride solution)

1

0.022

0.021

4

0.014

12

0.027

Positive control

(1%NaOH solution)

2

2.129

2.21

7

2.305

11

2.194

Test substance

(Tar, brown-coal, low-temp.)

5

1.110

1.00

10

0.948

15

0.943

 

IVIS (In Vitro Irritancy Score)

 

Application

substance

IVIS

Negative control

(0.9% sodium chloride solution)

0.65

Positive control

(1%NaOH solution)

139.51*

Test substance

(Tar, brown-coal, low-temp.)

27.03

* IVIS >55.1 – positive response

 

Applicant's summary and conclusion

Interpretation of results:
other: The substance is not defined as a corrosive or severe irritant
Remarks:
Criteria used for interpretation of results: EU
Executive summary:

The test was performed according to the OECD Test Guideline No. 437, Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Adopted 7th September 2009.

The BCOP test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and visible light spectrophotometer, respectively. Both measurements are used to calculate an IVIS (In Vitro Irritancy Score), which is used to assign an in vitro irritancy hazard classification category for prediction of an in vivo ocular irritation of the test substance.

The test was performed using nine isolated bovine corneas. Three corneas for each group - positive group, negative group and treatment group. Open-chamber method was used, because the substance is viscous liquid.

 

Results:

The In Vitro Irritancy Score (IVIS) is stated in the following table. IVIS of the test substance was found: 27.03. A substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

 

IVIS (In Vitro Irritancy Score)

  

Application

substance

IVIS


Negative control

(0.9% sodium chloride solution)

0.65

Positive control

(1%NaOH solution)

139.51*

Test substance

(Tar, brown-coal, low-temp.)

27.03

 

 

* IVIS >55.1 – positive response

 

The substance - Tar, brown-coal, low-temp - was not identified as an corrosive and severe irritant.