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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 conducted on 05 June 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identification: IFF TM 12-209
Storage Conditions: Approximately 4 °C in the dark
Physical state/Appearance: Extremely pale yellow liquid

Test animals / tissue source

Species:
other: Eyes from adult cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin and Streptomycin, and transported to the laboratory on ice packs. The corneas were prepared immediately on arrival.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
0.75 mL of test item was applied.
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
120 minutes
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Raeding
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test item and control items 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. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Results and discussion

In vivo

Results
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 120
Score:
7.1
Reversibility:
not specified
Irritant / corrosive response data:
Corneal Epithelium Condition
The condition of each cornea post treatment and at the final opacity measurement are provided. 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 virto Irritancy Score
The In Vitro irritancy scores are summarized as follows:
Test Item: 7.1
Negative Control: 0.6
Positive Control: 40.3

Any other information on results incl. tables

Criterion for an acceptable test

The positive control In Vitro Irritancy Score was within the range of 22.4 to 60.3. The positive control acceptance criterion was therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

Cornea No.

Opacity

Permeability (OD)

In vitro irritancy score

Pre-treatment

Post-treatment

Post-treatment – pre-treatment

Corrected value

 

Corrected value

Negative control

2

2

3

1

 

0.032

 

 

4

2

2

0

 

0.024

 

 

8

2

2

0

 

0.001

 

 

 

 

0.3*

 

0.019**

 

0.6

Positive control

1

3

23

20

19.7

1.343

1.324

 

3

3

25

22

21.7

1.201

1.182

 

7

3

26

23

22.7

1.313

1.294

 

 

 

 

 

21.3***

 

1.267***

40.3

Test item

5

1

9

8

707

0.101

0.082

 

6

3

12

9

8.7

0.068

0.049

 

9

2

5

3

2.7

0.041

0.022

 

 

 

 

 

6.3***

 

0.051***

7.1

OD = Optical density

* = Mean of the post-treatment-pre-treatment values

**= Mean permeability

***= Mean corrected value

 

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

2

Clear

Clear

4

Clear

Clear

8

Clear

Clear

Positive Control

1

Cloudy

Cloudy

3

Cloudy

Cloudy

7

Cloudy

cloudy

Test Item

5

Clear

Clear

6

Clear

Clear

9

clear

Clear

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant. The result of this study has identified the test item as not causing serious eye damage, but this does not permit conclusion that the test item does not require classification for eye irritation.
Executive summary:

The eye irritancy potential of the test substance, TM 12-209, was assessed as not irritating according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method.