4-amino-2-methylquinoline

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 11 to June 22, 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD guideline 438, EU Method B.48 and 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:

4-amino-2-methylquinoline

EC Number:

229-604-4

EC Name:

4-amino-2-methylquinoline

Cas Number:

6628-04-2

Molecular formula:

C10H10N2

IUPAC Name:

4-amino-2-methylquinoline

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 4-aminoquinaldine
- Molecular formula (if other than submission substance): C10H10N2
- Physical state: slightly yellow powder.
- Analytical purity: 98.5%
- Lot/batch No.:9213/R.83
- Storage condition of test material: 15-25 ºC

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 moistered properties 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. Control: 0.03mL.

Duration of treatment / exposure:

10 seconds.
Positive control: Imidazole.
Negative control: Physiological Salt

Observation period (in vivo):

30, 75, 120, 180, and 240 minutes (± 5 minutes).

Number of animals or in vitro replicates:

3 eyeballs for group. 3 groups (1 treated and 2 controls)

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 seconds-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 may lead to corneal damage. According to the GHS the test item can be classified into Category 1 (3x ICE class IV) so a second run of nine eyeballs was not conducted.

Any other information on results incl. tables

Table 1a. Fluorescein retention.

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	3	3	3	3	3	3	0	0	0

Table 2a. Corneal opacity.

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	3	3	3	4	4	4	0	0	0
75	3	3	3	4	4	4	0	0	0
120	3	3	3	4	4	4	0	0	0
180	3	3	3	4	4	4	0	0	0
240	3	3	3	4	4	4	0	0	0

Table 3. Corneal swelling (%)

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	24.7	32.2	55.1	43.3	52.9	49.8	2.0	2.7	5.1
75	86.6	52.7	58.6	66.6	61.4	75.4	0.3	2.0	4.1
120	106.2	54.7	75.0	77.5	69.8	81.9	-0.7	0.0	1.0
180	106.5	67.8	77.4	78.5	70.8	84.4	-1.7	-0.7	2.0
240	107.9	74.5	81.8	81.6	72.9	84.3	-3.0	-1.7	0.0

Table 4. Gross evaluation of the treated corneas.

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

Applicant's summary and conclusion

Interpretation of results:

Category I

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

The overall in vitro irritancy classification indicate that 4-Aminoquinaldine may lead to corneal damage.

Executive summary:

The study was performed according to OECD Guideline 438 and EU Method B.48. GLP study. Chicken heads were collected from a slaughter house near the laboratory facility. The eyeballs were extracted and were assessed for potential damage by corneal opacity profusion and fluorescein retention at the beginning of the study. The test item and the positive control (imidazole) were applied in the amount of 0.03 g, whereas the item used in the negative control (physiologic salt) was applied in a volume of 0.03 mL, all for a 10 seconds time. Three eyeballs were used for the test item and three for each control item. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure. The corneas treated with the test and 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 time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. At the end, the eyeballs were fixed in a 4% solution of formaldehyde to conduct histopathological examinations. The mean fluorescein retention score for the eyeballs treated with the test item was equal to 3.0 (ICE class IV). The mean corneal opacity score for the eyeballs treated with the test item was equal to 3.0 (ICE class IV). The mean corneal swelling values for the test item were from 37.3 to 88.1 (ICE class IV). The 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. Histopathology revealed cell alteration in the cornea and detachment of the anterior corneal epithelium. According to these results it may be stated that the test item may lead to corneal damage. According to the GHS the test item can be classified into Category 1, so a second run of nine eyeballs was not conducted.