Indoline-2,3-dione

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2015 - 20 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 438. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Indoline-2,3-dione

EC Number:

202-077-8

EC Name:

Indoline-2,3-dione

Cas Number:

91-56-5

Molecular formula:

C8H5NO2

IUPAC Name:

2,3-dihydro-1H-indole-2,3-dione

Details on test material:

- Name of test material (as cited in study report): indoline-2,3-dione
- Physical state: orange, crystalline powder
- Analytical purity: 98.0%
- Lot/batch No.: MP1076.3
- Storage condition of test material: 5-35ºC.

Test animals / tissue source

Species:

other: chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Age at study initiation: 7 weeks old
- Weight at study initiation: from 1.5 to 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

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.03 g

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

Number of animals or in vitro replicates:

9 eyeballs (3 eyeballs per each three groups: one treated group and two control groups).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with 20 mL of physiological salt.
- Time after start of exposure: after the 10 second-exposure period.

MEASURED PARAMETERS
The corneas treated with the test item and the control items were evaluated 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:
Fluorescein 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 using an SP-100 pachymeter.

Gross evaluation
The aim of this evaluation was 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 vivo

Irritant / corrosive response data:

On the grounds of the study results described in this Report 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 Category, since the ICE Class combination of the 3 endpoints were 3xI (the first run) and 3xI (the second run).

Any other information on results incl. tables

Table 1a. Fluorescein retention- the first run.

observation after time t (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
0	0	0	0	0	0	0	0	0	0
30	0	0	1	3	3	3	0	0	0

Table 1b. Fluorescein retention- the second run.

observation after time t (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
0	0	0	0	0	0	0	0	0	0
30	0.5	0.5	0.5	3	3	3	0	0	0

Table 2a. Corneal opacity – the first run.

observation after time t (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
0	0	0	0	0	0	0	0	0	0
30	0	0	1	4	4	4	0	0	0
75	0	0	1	4	4	4	0	0	0
120	0	0	1	4	4	4	0	0	0
180	0	0	1	4	4	4	0	0	0
240	0	0	1	4	4	4	0	0	0

Table 2b. Corneal opacity – the second run.

observation after time t (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
0	0	0	0	0	0	0	0	0	0
30	0.5	0.5	0.5	4	4	4	0	0	0
75	0.5	0.5	0.5	4	4	4	0	0	0
120	0.5	0.5	0.5	4	4	4	0	0	0
180	0.5	0.5	0.5	4	4	4	0	0	0
240	0.5	0.5	0.5	4	4	4	0	0	0

Table 3a. Corneal swelling (%) – the first run.

observation after time t (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
0	-	-	-	-	-	-	-	-	-
30	2.6	3.0	2.6	34.3	39.2	37.6	0.7	1.6	1.3
75	2.3	3.6	2.6	52.8	58.5	57.8	1.3	0.6	0.7
120	1.7	2.6	2.3	61.1	65.1	64.7	0.3	-0.3	0.0
180	1.0	2.0	1.3	62.4	67.4	67.6	-1.3	-1.3	-1.3
240	-0.3	0.3	0.3	65.0	70.1	69.9	-3.9	-2.6	-3.0

Table 3b. Corneal swelling (%) – the second run.

observation after time t (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
0	-	-	-	-	-	-	-	-	-
30	3.7	5.7	1.0	34.2	38.5	35.7	0.7	0.7	1.0
75	2.3	5.1	0.0	49.8	56.9	53.4	1.7	1.3	0.3
120	1.0	3.4	-1.0	53.5	64.5	58.7	0.0	0.3	-0.3
180	0.0	1.7	-1.3	57.5	68.8	63.0	-3.7	-2.0	-2.0
240	-1.0	-0.3	-2.3	59.8	71.1	64.6	-4.4	-3.4	-3.6

Table 4a. Gross evaluation of the treated corneas - the first run.

observation after time t (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

Table 4b. Gross evaluation of the treated corneas - the second run.

observation after time t (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	N

NC = no changes

SIGNS = roughening of the corneal surface

Histopathological evaluation of the treated corneas - results

The first run:

The negative control corneas had a normal histological structure.

Histopathological examinations of the positive control corneas revealed: edema of the anterior corneal epithelium (eyeball no. 4); cells vacuolation of the anterior corneal epithelium (eyeballs no. 4 and 5); dissection of the anterior corneal epithelium (eyeballs no. 4 and 5); detachment and erosions of the anterior corneal epithelium (eyeballs no. 4, no. 5 and 6). These changes confirmed corrosive properties of imidazole.

Histopathological examinations of the corneas treated with the test item showed a normal histological structure.

The second run:

The negative control corneas had a normal histological structure.

Histopathological examinations of the positive control corneas revealed: detachment and erosions of the anterior corneal epithelium (eyeballs no. 4, no. 5 and 6); lack of posterior corneal epithelium (eyeballs no. 4) and erosions of posterior corneal epithelium (eyeballs no. 5 and 6). These changes confirmed corrosive properties of imidazole.

Histopathological examinations of the corneas treated with the test item showed a normal histological structure.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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 Category, since the ICE Class combination of the 3 endpoints were 3xI (the first run) and 3xI (the second run).

Executive summary:

The isolated chicken eye test (in vitro) was performed according to the OECD Guideline 428 and EU Method B.48, following the Principles of GLP. The test item and the positive control (imidazole) were applied in the amount of 0.03 g, whereas the negative control (physiological salt) was applied in a volume of 0.03 mL. The test item and controls were applied for 10 seconds to three eyeballs each, and then rinsed with 20 mL of physiological salt at ambient temperature.Next, the eyeballs in their holders were placed in the superfusion apparatus in the original upright position. The corneas treated with the test item and the control items were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse, and then fixed in a 4% solution of formaldehyde to conduct histopathological examinations. 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. The mean fluorescein retention score for the eyeballs treated with the test item was equal to 0.3 (ICE class I) and 0.5 (ICE class I) in the first and second run, respectively. The mean corneal opacity score for the eyeballs treated with the test item was equal to 0.3 (ICE class I) and 0.5 (ICE class I) in the first and second run, respectively. The mean corneal swelling values for the test item were from 0.1 to 2.7 (ICE class I) in the first run and from 0.1 to 3.5 (ICE class I) in the second run. These results can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations did not reveal any changes of the corneal surface and histopathological examinations showed a normal histological structure. On the grounds of the study results described in this Report 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 Category, since the ICE Class combination of the 3 endpoints were 3xI (the first run) and 3xI (the second run).