Registration Dossier

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 the 6 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Identification: Lyrame
Physical state/Appearance: yellow viscous liquid
Storage Conditions: approximately 4 °C in the dark, under nitrogen

Test animals / tissue source

Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
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/Streptomycin, and transported to the laboratory on ice packs. The corneas were prepared immediately on arrival.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3 cornea for test and contol items
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 70 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 readings
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 cornea. 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 Labtech LT-4500 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.


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 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 Irritancy 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.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
0.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid

Any other information on results incl. tables

Criterion for an acceptable test

The positive control In Vitro Irritancy Score was above the range of 27.2 to 53.4. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.

The negative control gave opacity of ≤2.8 and permeability ≤0.115. The negative control acceptance criteria were therefore satisfied.

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

2

3

3

4

1

 

0.006

 

 

4

3

3

4

1

 

0.005

 

 

5

3

3

5

2

 

0.005

 

 

 

 

 

 

1.3*

 

0.005#

 

1.4

Positive Control

1

2

25

31

29

27.7

2.2

2.195

 

3

4

24

32

28

26.7

2.145

2.142

 

6

2

28

33

31

29.7

1.465

1.460

 

 

 

 

 

 

28.0~

 

1.931~

57.0

Test Item

7

4

3

5

1

0

0.036

0.031

 

8

2

2

4

2

0.7

0.009

0.004

 

9

3

4

4

1

0.0

0.006

0.001

 

 

 

 

 

 

0.2~

 

0.012~

0.4


OD= Optical density            

* = Mean of the post-incubation -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

5

clear

clear

Positive Control

1

cloudy

cloudy

3

cloudy

cloudy

6

cloudy

cloudy

Test Item

7

clear+

clear

8

clear+

clear

9

clear+

clear

+= small amount of test item adhered to the cornea.

Applicant's summary and conclusion

Interpretation of results:
other: Non irritant
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item, lyrame was considered not to be an ocular corrosive or severe irritant.
Executive summary:

The eye irritancy potential of the test item lyrame was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and has an IVIS of 0.4, according to EU CLP criteria.

According to OECD 437 the test item has no category. Not requiring classification to UN GHS or EU CLP.