Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 October 2014 - 24 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): 4-amino-5-(ethylsulphonyl)-o-anisic acid
- Physical state: Solid
- Analytical purity: 99.81%
- Lot/batch No.: MP1032.31

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Source: Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Age at study initiation: 7-week-old
- Weight at study initiation: 1.5 - 2.5 kg

BIOLOGICAL MATERIAL
After sedation of the chickens by electric shock and incision of the neck for bleeding, their heads were transported to the laboratory in a plastic container at
ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container.
The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g

Application and removal of the test item and the control items:
There were three groups, i.e. one group treated with the test item and two control groups, including a positive one (imidazole) and a negative one (physiological
salt) used concurrently in the study to ensure its quality. The test item and the control items were tested on three eyeballs each. Immediately following the zero
reference measurements, the eyeballs in their holders were removed from the superfusion apparatus and placed in a horizontal position in order to apply the test
item and the control items. The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g,
whereas the item used in the negative control (physiological salt) was applied in a volume of 0.03 mL. The test item and the control items were uniformly applied
to the corneal surface for 10 seconds. Then, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature.
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
4 hours after post-treatment rinse
Number of animals or in vitro replicates:
9 eyebalss (3 eyeballs per treatment: test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes (physiological salt)
- Time after start of exposure: After 10 seconds of exposure.

MEASURED PARAMETERS:
Pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
At all observation times points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The
quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure.

Scoring system
Florescein retention:
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

Corneal opacity:
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

Corneal swelling:
The degree of corneal swelling was determined by measuring corneal thickness with a SP-100 pachymeter.

Gross evaluation:
To determine whether any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the
cornea were visible.

Histopathological evaluation of the treated corneas:
Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4%
solution of formaldehyde. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was
dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using
Hematoxylin and Eosin. The following layers of the cornea were evaluated: anterior epithelium, anterior elastic lamina (Bowman’s membrane), corneal stroma,
posterior elastic lamina (Descemet’s membrane), and posterior epithelium. All treated eyeballs were subject to this evaluation.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
fluorescein retention score
Run / experiment:
MEAN, 1 run
Value:
ca. 1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
ICE class II
Irritation parameter:
fluorescein retention score
Run / experiment:
MEAN, RUN 2
Value:
ca. 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
ICE class III
Irritation parameter:
cornea opacity score
Run / experiment:
MEAN, RUN 1
Value:
ca. 1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
ICE class II
Irritation parameter:
cornea opacity score
Run / experiment:
MEAN, RUN 2
Value:
ca. 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
ICE class III
Irritation parameter:
percent corneal swelling
Run / experiment:
MEAN, RUN 1
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
ICE class I
Irritation parameter:
percent corneal swelling
Run / experiment:
MEAN, RUN 2
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
ICE class I
Irritation parameter:
morphological effects
Remarks on result:
not measured/tested
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:yes (SEE BELOW)
- Acceptance criteria met for positive control:yes (SEE BELOW)

Fluorescein retention :RUN 1 and RUN 2 - The mean fluorescein retention values for the concurrent positive and negative controls were 3.0 (ICE class IV) for imidazole and 0.0 (ICE class I) for physiological salt
Corneal opacity :RUN 1 and RUN 2 - The mean corneal opacity values for the concurrent positive and negative controls were 4.0 (ICE class IV) for imidazole and 0.0 (ICE class I) for physiological salt
Corneal swelling : FIRST RUN:The mean corneal swelling values for the positive control (imidazole) were from 56.7 to 99.2 (ICE class IV). As for the concurrent negative control samples (physiological salt), no swelling was observed (ICE class I).
: SECOND RUN: The mean corneal swelling values for the positive control (imidazole) were from 45.6 to 78.9 (ICE class IV). As for the concurrent negative control samples (physiological salt), no swelling was observed (ICE class I)

These values fell within the acceptable ranges.

In vivo

Irritant / corrosive response data:
On the grounds of the study and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run).
Other effects:
- Lesions and clinical observations: NO
- Ophthalmoscopic findings:NO
- Gross evaluation The negative control eyeballs and the ones treated with the test item did not exhibit any changes of the corneal surface (the first and the second run).

- Histopathological findings:
The first run - The negative control corneas had a normal histological structure. Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal epithelium in all eyeballs (eyeballs no. 4, 5, and 6). These changes confirmed corrosive properties of imidazole.Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal epithelium (eyeballs no. 2). The corneas had a normal histological structure in eyeball no 1 and no. 3.
The second run -The negative control corneas had a normal histological structure.Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal epithelium (eyeball no. 4, no. 5 and no. 6.); cell vacuolation and detachment of the posterior corneal epithelium (eyeball no. 4). These changes confirmed corrosive properties of imidazole. Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal epithelium in all eyeballs (eyeballs no. 1, no. 2, and no. 3).
- Effects of rinsing or washing: NO

Any other information on results incl. tables

RESULTS:

Evaluation of fluorescein retention– the first run.

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

1.3

II

3.0

IV

0.0

I

 

Evaluation of fluorescein retention – the second run.

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

2.0

III

3.0

IV

0.0

I

 

Evaluation of corneal opacity– the first run.

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

1.3

II

4.0

IV

0.0

I

75

1.3

II

4.0

IV

0.0

I

120

1.3

II

4.0

IV

0.0

I

180

1.3

II

4.0

IV

0.0

I

240

1.3

II

4.0

IV

0.0

I

 

Evaluation of corneal opacity – the second run.

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

2.0

III

4.0

IV

0.0

I

75

2.0

III

4.0

IV

0.0

I

120

2.0

III

4.0

IV

0.0

I

180

2.0

III

4.0

IV

0.0

I

240

2.0

III

4.0

IV

0.0

I

 

Evaluation of corneal swelling (%) – the first run.

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

0.9*

I

56.7

IV

1.5*

I

75

2.5*

I

66.8

IV

2.5*

I

120

4.5*

I

84.4

IV

4.3*

I

180

5.8*

I

93.1

IV

5.9*

I

240

7.8*

I

99.2

IV

6.8*

I

* - percentage of corneal thickness decrease, no swelling

 

Evaluation of corneal swelling (%) – the second run.

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Average

ICE class

Average

ICE class

Average

ICE class

30

0.3*

I

45.6

IV

1.4*

I

75

1.0*

I

53.4

IV

2.4*

I

120

3.1*

I

64.5

IV

4.5*

I

180

5.7*

I

75.0

IV

5.3*

I

240

6.6*

I

78.9

IV

6.5*

I

* - percentage of corneal thickness decrease, no swelling

 Fluorescein retention- the first run.

observation after

time t

(minutes)

test item

 

 

eyeball no.

positive control

imidazole

 

eyeball no.

negative control

physiological saline

 

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

0

0

0

0

0

0

0

0

0

30

1

2

1

3

3

3

0

0

0

 Fluorescein retention- the second run.

observation after

time t

(minutes)

test item

 

 

eyeball no.

positive control

imidazole

 

eyeball no.

negative control

physiological saline

 

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

0

0

0

0

0

0

0

0

0

30

2

2

2

3

3

3

0

0

0

Corneal capacity:first run

observation after

time t

(minutes)

test item

 

 

eyeball no.

positive control

imidazole

 

eyeball no.

negative control

physiological saline

 

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

0

0

0

0

0

0

0

0

0

30

1

2

1

4

4

4

0

0

0

75

1

2

1

4

4

4

0

0

0

120

1

2

1

4

4

4

0

0

0

180

1

2

1

4

4

4

0

0

0

240

1

2

1

4

4

4

0

0

0

Corneal capacity:second run

observation after

time t

(minutes)

test item

 

 

eyeball no.

positive control

imidazole

 

eyeball no.

negative control

physiological saline

 

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

0

0

0

0

0

0

0

0

0

30

2

2

2

4

4

4

0

0

0

75

2

2

2

4

4

4

0

0

0

120

2

2

2

4

4

4

0

0

0

180

2

2

2

4

4

4

0

0

0

240

2

2

2

4

4

4

0

0

0

Corneal swelling (%) – the first run

observation after

time t

(minutes)

test item

 

 

eyeball no.

positive control

imidazole

 

eyeball no.

negative control

physiological saline

 

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

-

-

-

-

-

-

-

-

-

30

-1.0

-0.7

-1.0

50.8

56.6

62.7

-0.7

-1.0

-2.78

75

-2.0

-2.1

-3.4

60.9

69.2

70.2

-1.0

-1.7

-4.7

120

-4.4

-4.1

-5.1

79.9

83.4

89.8

-2.0

-5.3

-5.7

180

-5.1

-5.8

-6.4

87.3

92.7

99.3

-4.0

-6.6

7.0

240

-6.8

-7.9

-8.8

93.6

99.0

105.1

-5.1

-7.3

-8.1

Corneal swelling (%) – the second run

observation after

time t

(minutes)

test item

 

 

eyeball no.

positive control

imidazole

 

eyeball no.

negative control

physiological saline

 

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

-

-

-

-

-

-

-

-

-

30

-0.9

0.3

-0.3

45.4

46.6

44.8

-1.2

-0.6

-2.3

75

-1.5

-0.6

-0.9

56.3

52.3

51.5

-2.4

-1.4

-3.4

120

-5.9

-1.8

-1.5

64.1

60.9

68.6

-6.5

-2.8

-4.3

180

-6.2

-5.9

-5.0

73.3

76.6

75.0

-7.1

-3.4

-5.4

240

-7.1

-6.2

-6.4

78.2

80.9

77.6

-8.2

-4.3

-6.9

Gross evaluation of the treated corneas - the first run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Eyeball no.

Eyeball no.

Eyeball no.

1

2

3

4

5

6

7

8

9

30

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

75

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

120

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

180

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

240

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

NC = no changes

SIGNS = roughening of the corneal surface

 

Gross evaluation of the treated corneas - the second run.

 

Observation after (minutes)

Test item

Positive control

Imidazole

Negative control

Physiological saline

Eyeball no.

Eyeball no.

Eyeball no.

1

2

3

4

5

6

7

8

9

30

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

75

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

120

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

180

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

240

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

NC = no changes

SIGNS = roughening of the corneal Surface

Histological examination of the corneas treated with the test item - the first run.

The negative control corneas had a normal histological structure.

Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal

epithelium in all eyeballs (eyeballs no. 4, 5, and 6). These changes confirmed corrosive properties of imidazole.

Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal

epithelium (eyeballs no. 2). The corneas had a normal histological structure in eyeball no 1 and no. 3.

Histopathological examination of the corneas treated with the test item - the second run.

The negative control corneas had a normal histological structure.

Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal

epithelium (eyeball no. 4, no. 5 and no. 6.); cell vacuolation and detachment of the posterior corneal epithelium

(eyeball no. 4). These changes confirmed corrosive properties of imidazole.

Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal

epithelium in all eyeballs (eyeballs no. 1, no. 2, and no. 3).

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run).
Conclusions:
The test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made.
Executive summary:

The isolated chicken eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48. The study was conducted in two runs. The first study led to a GHS NC outcome, so a second run of nine eyeballs was conducted to confirm or discard the negative outcome. The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g, whereas the item used in the negative control (physiological salt) was applied in a volume of 0.03 mL. Three eyeballs were used for the test item and three for each control item. Every time, the test item and the control items were applied to the corneal surface for 10 seconds and kept at temperature between 20 – 23º C. Then, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature. The corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure. Following the final evaluation of the treated eyeballs, i.e. 240 minutes after the application of the test item and the control items, the eyeballs were fixed in a 4% solution of formaldehyde in order to allow histopathological examinations to be conducted. The test item did not cause eye damage. The mean fluorescein retention scores for the eyeball treated with test item were 1.3 (ICE class II) and 2.0 (ICE class III) in the first and second round respectively. The mean corneal opacity scores were 1.3 (ICE class II) and 2.0 (ICE class III). No swelling was observed in both rounds (ICE class I). Gross examination did not reveal any changes of the corneal surface. The histopathological examinations of the corneas showed exfoliation of the anterior corneal epithelium in one eye in the first run and in all three eyes in the second one. These results were accepted since the concurrent positive and negative control values fell within the acceptable ranges for the method. On the grounds of the study results and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run).