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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:
2010-03-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and conducted to guideline

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The neat test material was applied for 240 minutes. Negative and positive control materials 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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Molecular formula : Mn(NO3)2.4H2O
- Physical state: solid
- Storage condition of test material: in the dark at 4°C

Test animals / tissue source

Species:
other: Adult Bovine eyes sourced from local abattoir as a by-product of freshly slaughtered animals.
Strain:
other: Cattle
Details on test animals or tissues and environmental conditions:
Not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: no control animals were used, but three bovine eyes were used for both the positive and negative controls
Amount / concentration applied:
Not reported
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
No animals were used, but three bovine eyes were used.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period the neat test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM.
- Time after start of exposure: 4 hours

SCORING SYSTEM:

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 corneaThe 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 material induced a response through only one of the two endpoints


TOOL USED TO ASSESS SCORE: fluorescein

Results and discussion

In vivo

Results
Irritation parameter:
overall irritation score
Basis:
other: mean opacity value + (15 x mean OD492 value)
Time point:
other: 4 hours
Score:
82.7
Reversibility:
not specified
Irritant / corrosive response data:
Not reported
Other effects:
Not reported

Any other information on results incl. tables

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 OD492was calculated by subtracting the mean OD492of the negative control corneas from the OD492value of each treated cornea. The OD492value of each treatment group was calculated by averaging the corrected OD492values of the treated corneas for the treatment group.

In vitro irritancy score:

The following formula was used to determine the in vitro score:

           In VitroIrritancy Score = mean opacity value + (15 x mean OD492value)

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.

Visual observation

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

Data interpretation

A test material that induces an in vitro irritancy score >= 55.1 is defined as an ocular corrosive or severe irritant.

                  

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 is given in Table 2.

The corneas treated with the test material or positive control material were cloudy post treatment. The corneas treated with the negative control material were clear post treatment.

 In Vitro Irritancy Score

The results are summarised as follows:

Treatment

In Vitro Irritancy Score

Classification

Test Material

82.7

severe irritant

Negative Control

1.5

mild irritant

Positive Control

119.8

severe irritant

Table1              Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In vitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

1

2

1

 

0.026

 

 

2

2

3

1

 

0.027

 

 

3

1

2

1

 

0.039

 

 

 

 

 

1.0*

 

0.031¨

 

1.5

Positive Control

7

1

74

73

72.0

3.425

3.394

 

8

1

74

73

72.0

2.490

2.459

 

9

1

71

70

69.0

3.940

3.909

 

 

 

 

 

71.0·

 

3.254·

119.8

Test Material

4

1

76

75

74.0

0.336

0.305

 

5

1

80

79

78.0

0.303

0.242

 

6

2

87

85

84.0

0.257

0.226

 

 

 

 

 

78.7·

 

0.268·

82.7


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

Table2              Corneal Epithelium Condition

Treatment

Cornea Number

Observation

Immediately after Rinsing

Negative Control

1

clear

2

clear

3

clear

Positive Control

7

cloudy

8

cloudy

9

cloudy

Test Material

4

cloudy

5

cloudy

6

cloudy

Applicant's summary and conclusion

Interpretation of results:
corrosive
Conclusions:
The test material is corrosive to the eyes and requires classification as R41 and Cat 1 eye irritant.