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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:
The study was performed on 11 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection : 10 July 2012 Date of Signature : 05 February 2013

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Sponsor's identification : Amyl Salicylate
CAS number : 2050-08-0
Description : clear colourless liquid
Batch number : PE00022143
Purity : 99.99%
Date received : 03 October 2011
Expiry date : 19 August 2012
Storage conditions : room temperature in the dark

This study was scheduled to be conducted after the test item expiry date of 19 August 2012. The sponsor was therefore contacted prior to commencement of the study. The sponsor tested the test item in order to re-validate the quality of the batch with the conclusion that the quality of the batch is comparable to the specification given in the test item Technical Data Sheet originally supplied with the test item. This study therefore proceeded as scheduled.

Test animals / tissue source

Species:
cattle
Strain:
other: bovine cornea from slaughterhouse
Details on test animals or tissues and environmental conditions:
Not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST ITEM

-Amounts(s) applied (volume or weight with unit):
0.75 mL of the test item was applied to triplicate corneas.

-Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes.
Duration of post- treatment incubation (in vitro):
Post-treatment incubation period of 120 minutes.
Number of animals or in vitro replicates:
triplicates for test item, positive and negative control.
Details on study design:
TEST SITE
-Area of exposure
0.75 mL of the test item was applied to triplicate corneas.

-% coverage:
The test item was topically applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.

-Type of wrap used:
None used

REMOVAL OF TEST ITEM
-Washing (if done):
At the end of the exposure period the test item was removed from the anterior chamber and each cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenaol red.

-Time after start of exposure:
10 minutes post exposure

EVALUATION OF RESULTS
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement-
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the in vitro score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

DATA INTERPRETATION
A test item that induces an in vitro irritancy score greater than or equal to 55.1 is defined as an ocular corrosive or severe irritant.



Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
test item
Value:
2.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item induced an in vitro irritancy score of 2.7.
The corneas treated with the test item were clear post treatment and post incubation.

Any other information on results incl. tables

RESULTS

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

Corneal Epithelium Condition

The condition of the cornea immediately after rinsing and at the final opacity measurement is given in Table 2.

The corneas treated with the test item were clear 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.

In Vitro Irritancy Score

The results are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Item

2.7

Negative Control

2.7

Positive Control

36.1

Criteria for an Acceptable Test

The positive control In Vitro irritancy Score was within the range of 30.9 to 67.7. The positive control acceptance criterion was therefore satisfied.

Table 1          Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In vitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Incubation

Post-Incubation-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

4

5

8

4

 

0.051

 

 

2

3

2

4

1

 

0.046

 

 

3

2

2

3

1

 

0.033

 

 

 

 

 

 

2.0*

 

0.043+

 

2.7

Positive Control

4

2

31

27

25

23.0

1.155

1.112

 

5

2

30

25

25

23.0

0.872

0.829

 

6

1

26

24

24

22.0

0.792

0.749

 

 

 

 

 

 

22.7·

 

0.896·

36.1

Test Item

7

2

5

7

5

3.0

0.019

0.0

 

8

1

4

6

5

3.0

0.020

0.0

 

9

2

5

6

4

2.0

0.034

0.0

 

 

 

 

 

 

2.7·

 

0.0·

2.7


OD = Optical density  * = Mean of the post incubation-pre‑treatment values  + = Mean permeability       · = Mean corrected value


Table 2          Corneal Epithelium Condition

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

1

clear

clear

2

clear

clear

3

clear

clear

Positive Control

4

cloudy

cloudy

5

cloudy

cloudy

6

cloudy

cloudy

Test Item

7

clear

clear

8

clear

clear

9

clear

clear


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant.
Executive summary:

Introduction. A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 437 (2009) “Bovine Corneal Opacity and Permeability Assay”

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). 

Results. The in vitro Irritancy scores are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Item

2.7

Negative Control

2.7

Positive Control

36.1

Conclusion. The test item was considered not to be an ocular corrosive or severe irritant.