Indoline-2,3-dione

Endpoint summary

Description of key information

Skin irritation/corrosion: Key study: Test method according to OECD Guideline 430. GLP study. The test item does not lead to skin corrosion/severe irritation. 
Eye irritation: Test method according to OECD Guideline 438. GLP study. 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.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2015 - 19 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 430. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre for Experimental Medicine at the Medical University in Katowice
- Age at study initiation: 21 days old
- Weight at study initiation:
- Housing: plastic cage covered with a wire bar lid. The dimensions of the cage were 58 x 37 x 21 cm. UV-sterilized wood shavings were used as bedding.
- Diet (e.g. ad libitum): "Murigan" standard granulated laboratory fodder, ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23°C
- Humidity (%): 44 – 50%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Type of coverage:

other: Not applicable: in-vitro test

Preparation of test site:

shaved

Vehicle:

water

Controls:

yes, concurrent vehicle

Amount / concentration applied:

VEHICLE
- Amount(s) applied: 150 μL of distilled water.

Duration of treatment / exposure:

24 hours.

Number of animals:

2

Irritant / corrosive response data:

On the grounds of the study, it may be stated that the test item belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Table 1. Results of the control transcutaneous electrical resistance test (TER).

Animal number	Skin disc number	TER value (kΩ)
1	1	11.42
	2	13.11
2	1	16.52
	2	16.93

The skin discs gave the resistance values greater than 10 kΩ; therefore, the remainder of the skin discs of the animals could have been used in the experiment.

Table 2.Results of the control transcutaneous electrical resistance test (TER).

Animal number	Tested substance	Skin disc number	TER value (kΩ)	Mean TER value ± SD (kΩ)
1	Positive control – 36% HCl	1	0.88	0.88 ± 0.02
		2	0.86
		3	0.89
	Negative control – distilled water	1	19.23	19.30 ± 0.33
		2	19.01
		3	19.65
	Test item	1	19.10	19.05 ± 0.30
		2	18.73
		3	19.32
2	Positive control – 36% HCl	1	0.93	0.94 ± 0.01
		2	0.95
		3	0.94
	Negative control – distilled water	1	20.03	19.82 ± 0.19
		2	19.75
		3	19.67
	Test item	1	16.92	16.61 ± 0.28
		2	16.39
		3	16.52

The concurrent mean values for the positive and negative controls were within the acceptable ranges for the method:

Positive control: 0.5 – 1.0 kΩ

Negative control: 10 - 25 kΩ

Table 3. Gross changes on the surface of the treated skin discs.

Animal number	Tested substance	Skin disc number	Gross changes
1	Positive control – 36% HCl	1	Perforation
		2	Perforation
		3	Perforation
	Negative control – distilled water	1	No changes
		2	No changes
		3	No changes
	Test item	1	No changes
		2	No changes
		3	No changes
2	Positive control – 36% HCl	1	Perforation
		2	Perforation
		3	Perforation
	Negative control – distilled water	1	No changes
		2	No changes
		3	No changes
	Test item	1	No changes
		2	No changes
		3	No changes

The gross examination showed that the positive control skin discs exhibited skin perforation, whereas the negative control skin discs and the ones treated with the test item did not reveal any changes.

Interpretation of results:

other: non-corrosive/severe irritant.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The substance does not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to the OECD Guideline 430 and EU Method B.40, following the Principles of GLP. Skin discs used in the experiment were obtained from two 30-day-old animals. The test item (ground to a powder) was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Positive (36% hydrochloric acid) and negative (distilled water) controls were conducted concurrently. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water and the surface tension of the skin was reduced by adding 70% ethanol. After removing the ethanol the tissue was hydrated by the addition of 3 mL of a solution of MgSO₄ (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. The skin discs were subjected to a gross examination. The mean TER results for the skin discs treated with the test item were equal to 19.05 kΩ (animal no. 1) and 16.61 kΩ (animal no. 2). They can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs treated with the test item did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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.

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.

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).

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion: Key study: The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to the OECD Guideline 430 and EU Method B.40, following the Principles of GLP. The mean TER results for the skin discs treated with the test item were equal to 19.05 kΩ (animal no. 1) and 16.61 kΩ (animal no. 2). Gross examinations of the skin discs treated with the test item did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Eye irritation: Key study: 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 control items were applied to the corneal surface for 10 seconds. Then, they were rinsed from the eye 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. Corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed 30 minutes after the end of the exposure. For the treated eyeballs, the mean fluorescein retention score 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 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 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. Following the final evaluation of the treated eyeballs, gross examinations of the eyeballs did not reveal any changes of the corneal surface and histopathological examinations of the corneas showed a normal histological structure. On the grounds of the study results it may be stated that the test item did not cause eye damage. The test item is not classified 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).

Justification for selection of skin irritation / corrosion endpoint:
Only one study is available.

Justification for selection of eye irritation endpoint:
Only one study is available.

Justification for classification or non-classification

Based on the available data, the substance is not classified according to CLP Regulation (EC) no. 1272/2008.