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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

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

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to
Guideline:
other: The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM)
Deviations:
no
Qualifier:
according to
Guideline:
other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
Deviations:
no
Qualifier:
according to
Guideline:
other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: Liquid.
- Storage condition of test material: At room temperature protected from light.
- Other: Colourless

Test animals / tissue source

Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse.
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Housing: The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (recognised supplier) with the endothelial
side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Three corneas were used for the positive and negative control groups.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 microlitres
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 ±1 minutes
Observation period (in vivo):
After treatment the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.
Number of animals or in vitro replicates:
Three corneas were used for each treatment group
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After treatment with the test material the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.
- Time after start of exposure: 10 ± 1 minutes

SCORING SYSTEM:
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints. A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. A IVIS score of less than 3 is interpreted as having no prediction for corrosive or irritant effects.

TOOL USED TO ASSESS SCORE: Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
10 minutes exposure
Run / experiment:
mean
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
IVIS scores ranged from -0.1 to 2 after 10 minutes treatment
Other effects / acceptance of results:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 128.2 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.3 after 10 minutes of treatment. Additionally, all opacity and permeability responses were within the ranges of the historic negative controls.

No pH effect of the test substance was observed on the rinsing medium.

Any other information on results incl. tables

Table 1 Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity #1

Mean Permeability #1

Mean In vitro Irritation Score #1 , 2

Negative Control

-0.3

0.000

0.0

Positive Control

(Benzalkonium chloride)

81.7

3.102

128.2

Test substance

1.3

-0.004

1.3

#1 Calculated using the negative control mean opacity and mean permeability values

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2 Opacity score

Eye

Opacity before treatment

Opacity after treatment

Final opacity #1

Negative control corrected final opacity #2

Mean Opacity

Negative Control

1

1

1

0

0.0

0.0

2

0

0

0

0.0

3

0

0

0.0

0.0

Positive Control

4

-1

83

84

84.0

81.7

5

-1

83

84

84.0

6

-1

76

77

77.0

Test substance

7

-2

0

2

2.0

1.3

8

0

0

0

0.0

9

0

2

2

2.0

#1 Final Opacity = Opacity after treatment – Opacity before treatment

#2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

 

Table 3 Permeability score individual values (uncorrected)

Eye

Dilution Factor

OD490 1

OD490 2

OD490 3

Average OD490

Final OD

Mean Final Negative Control

 

Negative Control

 

1

1

0.010

0.011

0.011

0.011

0.011

0.008

2

1

0.004

0.004

0.004

0.004

0.004

3

1

0.009

0.007

0.008

0.003

0.008

 

Positive Control

 

4

6

0.314

0.311

0.312

0.312

1.874

5

6

0.482

0.479

0.473

0.478

2.868

6

6

0.794

0.781

0.775

0.783

4.700

 

Test substance

7

1

0.003

0.002

0.003

0.003

0.003

8

1

0.001

0.002

0.001

0.001

0.001

9

1

0.010

0.006

0.008

0.008

0.008

 

Table 4 In vitro irritancy score

Eye

Negative control corrected final opacity

Negative control corrected Final OD490

In vitro Irritancy Score #1

Negative Control

1

0.0

0.003

0.0

2

0.0

-0.004

-0.1

3

0.0

0.000

0.0

Positive Control

4

84.0

1.829

111.4

5

84.0

2.823

126.3

6

77.0

4.655

146.8

Test substance

7

2.0

-0.005

1.9

8

0.0

-0.006

-0.1

9

2.0

0.000

2.0

#1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions the test material is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test.
Executive summary:

The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test material to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the test material was placed the cornea. The negative control group received saline and the positive control group received 10% benzalkonium chloride. For each group the corneas were incubated for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 128.2 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.3 after 10 minutes of treatment. All opacity and permeability responses were within the ranges of the historic negative controls. Based on these results the test material is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.