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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation
Remarks:
other: ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed on 04 February 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 437
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Sponsor's identification : Benzyl Salicylate
Description : Clear colourless liquid
Batch number : PE00016783
Purity : 99.7%
Date received : 03 October 2011
Expiry date : 02 June 2012
Storage conditions : Room temperature in the dark

Test animals / tissue source

Species:
other: Excised Bovine Cornea
Strain:
other: Not Applicable
Details on test animals or tissues and environmental conditions:
Not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL

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

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

VEHICLE
No vehicle used
Duration of treatment / exposure:
The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes.
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
TEST SITE
-Area of exposure
0.75 mL of the test material was applied to triplicate corneas.

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

-Type of wrap used:
None used

REMOVAL OF TEST SUBSTANCE
-Washing (if done):
At the end of the exposure period the test material 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.

-Time after start of exposure:
10 minutes post exposure

SCORING SYSTEM:
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.

The following formula was used to determine the in vitro score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

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

Additionally, the opacity and permeability values were evaluated independently to determine whether the test material 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.

Results and discussion

In vivo

Results
Irritation parameter:
cornea opacity score
Basis:
other: Mean Score of Opacity & Permeability
Time point:
other: 120 Minutes Post Rinsing
Max. score:
0
Reversibility:
other:
Remarks on result:
other: See Below
Irritant / corrosive response data:
The test material induced an in vitro irritancy score of 0.0
Other effects:
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.

In Vitro Irritancy Score

The results are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Material

0.0

Negative Control

3.8

Positive Control

31.2

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

3

3

6

3

 

0.036

 

 

2

2

2

5

3

 

0.031

 

 

3

2

2

6

4

 

0.025

 

 

 

 

 

 

3.3*

 

0.031¨

 

3.8

Positive Control

4

3

26

27

24

20.7

0.698

0.667

 

5

2

27

27

25

21.7

0.614

0.583

 

6

4

31

31

27

23.7

0.610

0.579

 

 

 

 

 

 

22.0·

 

0.610·

31.2

Test Item

10

2

3

3

1

0.0

0.033

0.002

 

11

3

3

4

1

0.0

0.015

0.000

 

12

3

3

3

0

0.0

0.015

0.000

 

 

 

 

 

 

0.0·

 

0.001·

0.0


OD= Optical density      * = Mean of the post treatment-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

10

clear

clear

11

clear

clear

12

clear

clear


Applicant's summary and conclusion

Interpretation of results:
other: Non-Corrosive
Remarks:
Criteria used for interpretation of results: expert judgment
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant, in vitro.
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

0.0

Negative Control

3.8

Positive Control

31.2

Conclusion. 

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