1-Piperazineacetamide, N-(2,6-dimethylphenyl)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2011 - 19 November 2011

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:

2014

Report date:

2014

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:

N-(2,6-dimethylphenyl)-2-(piperazin-1-yl)acetamide

EC Number:

610-916-8

Cas Number:

5294-61-1

Molecular formula:

C14H21N3O

IUPAC Name:

N-(2,6-dimethylphenyl)-2-(piperazin-1-yl)acetamide

Details on test material:

- Name of test material (as cited in study report): N-(2,6-dimethylphenyl)-1-piperazineacetamide
- Physical state: solid
- Analytical purity: 99.8%
- Lot/batch No.: M13856C
- Expiration date of the lot/batch: December 2015
- Storage condition of test material: room temperature, i.e. 15-30ºC.

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: 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 a 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 (volume or weight with unit): 0.03 g.

Duration of treatment / exposure:

10 seconds.

Observation period (in vivo):

4 hours.

Number of animals or in vitro replicates:

9 eyeballs: 3 eyeballs per group (test item, positive control and negative control).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes (physiological salt)
- Time after start of exposure: 10 seconds.

MEASURED PARAMETERS: the corneas treated with the test item and the control items were evaluated pre-treatment and starting at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation times points, corneal opacity, corneal swelling, and morphological changes of the corneal surface were recorded. Fluorescein retention was determined once, 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:
Corneal swelling was determined by measuring corneal thickness with the SP-100 pachymeter (TOMEY).

Gross examination of the treated corneas:
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 examinations 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% formaldehyde solution. Next, one slice from each eyeball covering the cornea with the surrounding sclera was collected. The tissue material was dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness were 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.

Results and discussion

In vivo

Irritant / corrosive response data:

On the grounds of the study results and the overall in vitro irritancy classification, it may be stated that the test item can cause eye damage. According to the UN GHS classification criteria, the test item can be classified into Category 1, since the ICE class combination of the 3 endpoints was 3xIV (the first run) and 3xIV (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)
	Average	ICE class	Average	ICE class	Average	ICE class
30	3.0	IV	3.0	IV	0.0	I

Table 1b. Fluorescein retention - the second run.

Observation after time t (minutes)	Test item		Positive control (imidazole)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	3.0	IV	3.0	IV	0.0	I

Table 2a. Corneal opacity - the first run.

Observation after time t (minutes)	Test item		Positive control (imidazole)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	3.0	IV	4.0	IV	0.0	I
75	3.0	IV	4.0	IV	0.0	I
120	3.0	IV	4.0	IV	0.0	I
180	3.0	IV	4.0	IV	0.0	I
240	2.3	III	4.0	IV	0.0	I

Table 2b. Corneal opacity - the second run.

Observation after time t (minutes)	Test item		Positive control (imidazole)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	3.0	IV	4.0	IV	0.0	I
75	3.0	IV	4.0	IV	0.0	I
120	3.0	IV	4.0	IV	0.0	I
180	2.7	IV	4.0	IV	0.0	I
240	2.3	III	4.0	IV	0.0	I

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

Observation after time t (minutes)	Test item		Positive control (imidazole)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	44.30	IV	81.19	IV	1.53*	I
75	28.47	IV	78.24	IV	2.67*	I
120	52.05	IV	87.44	IV	1.58*	I
180	58.52	IV	88.52	IV	1.84*	I
240	64.95	IV	72.66	IV	2.62*	I

 “-“ = decrease in corneal thickness, no swelling.

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

Observation after time t (minutes)	Test item		Positive control (imidazole)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	34.95	IV	84.91	IV	0.13*	I
75	29.33	IV	98.70	IV	1.85*	I
120	32.37	IV	96.63	IV	3.85*	I
180	54.28	IV	107.89	IV	5.84*	I
240	50.43	IV	107.15	IV	7.29*	I

 “-“ = decrease in corneal thickness, no swelling.

Table 4a. Gross examinations 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	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
75	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
120	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
180	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
240	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC

NC = no changes

SIGNS = roughening of the corneal surface

Table 4b. Gross examinations 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	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
75	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
120	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
180	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
240	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC

NC = no changes

SIGNS = roughening of the corneal surface

Histopathological examinations of the corneas:

First run: The negative control corneas had a normal histological structure. The only exception was eyeball no. 8 in which exfoliation of the anterior corneal epithelium was stated. Histopathological examinations of the positive control corneas revealed defects of the anterior corneal epithelium (eyeballs no. 4, 5, and 6) and detachment of the anterior corneal epithelium (eyeball no. 5). These changes confirmed corrosive properties of imidazole. Histopathological examinations of the corneas treated with the test item showed dissection of the anterior corneal epithelium in all eyeballs (eyeballs no. 1, 2, and 3), defects of the anterior corneal epithelium (eyeballs no. 1 and 3), and necrosis of the anterior corneal epithelium (eyeball no. 3).

Second run: The negative control corneas had a normal histological structure. Histopathological examinations of the positive control corneas revealed defects of the anterior corneal epithelium (eyeballs no. 4, 5 and 6), detachment of the anterior corneal epithelium (eyeball no. 5), dissection of the anterior corneal epithelium (eyeballs no. 4 and 6), and cell vacuolation of the anterior corneal epithelium (eyeball no. 6). These changes confirmed corrosive properties of imidazole.

Histopathological examinations of the corneas treated with the test item showed dissection of the anterior corneal epithelium (eyeballs no. 1, 2, and 3), defects of the anterior corneal epithelium (eyeballs no. 2 and 3), and cell vacuolation of the anterior corneal epithelium (eyeball no. 2).

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Migrated information

Conclusions:

The test item can cause eye damage. According to UN GHS classification criteria, the test item can be classified into Category 1, since the ICE class combination of the 3 endpoints was 3xIV (the first run) and 3xIV (the second run).

Executive summary:

The isolated chicken eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48 (GLP study). Toxic effects to the cornea were measured by a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention), a qualitative assessment of opacity, a quantitative measurement of increased thickness (swelling), and macroscopic and histopathological examinations of morphological damage to the surface. The study was conducted in two runs. 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 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 treated with the test item and the control items were evaluated pre-treatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation time points, corneal opacity, corneal swelling, and morphological changes of the corneal surface were recorded. Fluorescein retention was determined 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 caused eye damage. For the eyeballs treated with the test item, the mean fluorescein retention scores were 3.0 (ICE class IV) in both first and second runs. The mean corneal opacity scores ranged from 2.3 (ICE class III) to 3.0 (ICE class IV) in both rounds. The mean corneal swelling values ranged from 28.47 (ICE class IV) to 64.95 (ICE class IV) in the first run, and from 29.33 (ICE class IV) to 54.28 (ICE class IV) 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 of the eyeballs treated with the test item revealed roughening of the corneal surface in both first and second run. Histopathological examinations of the corneas showed dissection of the anterior corneal epithelium and defects on the anterior corneal epithelium in both runs in addition to necrosis of the anterior corneal epithelium in the first run and cell vacuolation of the anterior corneal epithelium in the second run. According to the UN GHS classification criteria, the test item can be classified into Category 1, since the ICE class combination of the 3 endpoints was 3xIV (the first run) and 3xIV (the second run).