N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept - Oct 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

EC Number:

260-125-3

EC Name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Cas Number:

56358-10-2

Molecular formula:

C32H37N5

IUPAC Name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Test material form:

solid

Details on test material:

Batch number ZD 00883/044. Manufactured Oct 1996
91.3% purity. Impurity is residual solvent Shellsol AB

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr K. Thomae GMBH, Biberach
- Age at study initiation: Young Adults
- Weight at study initiation: 3.5kg - 3.97kg
Animal 1: 3.50kg (Male)
Animal 2: 3.95 kg (Male)
Animal 3: 3.97kg (Male)
- Housing: 1 animal per cage. Stainless steel wire mesh. Floor area 3000cm2. No bedding in the cages, sawdust in waste trays.
- Diet: Kliba-Labordiaet, Klingentalmuehle AG, Switzerland. About 350g per animal per day.
- Water: About 250mL tap water per animal per day
- Acclimation period: At least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Test system

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5ml applied in a patch 2.5cm x 2.5cm. The substance was heated to 90C and cooled to body temperature before application,

Duration of treatment / exposure:

4 hours

Observation period:

15 days. After the 4 hour exposure period, the test substance was removed with a Polyethylene glycol/water solution. Oberservations were made at the following intervals after removal of the patch:
1 hour
24 hours
48 hours
72 hours
8 days
15 days
A check for dead animals was made at least once a day.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: Upper third of the back or flanks
- 0.5mls of substance, covering an area 2.5cm x 2.5cm
- Type of wrap: Semiocclusive; Idealbinde.

REMOVAL OF TEST SUBSTANCE
- Washing: With a Polyethylene Glycol (Lutrol E400) and water mix (2:1)
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1 hour
24 hours
48 hours
72 hours
8 days
15 days

In Animal 1, a cross incision of the treated skin was peformed after the animal was killed at study termination. Additional histopathological examination was performed.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

2.7

Max. score:

4

Reversibility:

not reversible

Remarks:

within 15 days

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

erythema score

Remarks on result:

not determinable

Remarks:

Could not be read because of staining due to the colour of the test substance

Any other information on results incl. tables

Individual observations taken for each animal are tabulated below:

Time of observation	Animal number	Erythema score	Edema score	Comments on symptoms
1 hour	1	-	3	Index for Erythema could not be read because of staining due to the colour of the test substance
	2	-	2
	3	-	1
24 hours	1	-	4	Index for Erythema could not be read because of staining due to the colour of the test substance
	2	-	4	Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 3
	3	-	2
48 hours	1	-	3	Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 2
	2	-	3
	3	-	2
72 hours	1	-	3	Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 2
	2	-	2	Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 1
	3	-	1	Index for Erythema could not be read because of staining due to the colour of the test substance. Index for Erythema of the surrounding area: 1
8 days	1	0	2	Superficial scabbing. Application area reddish discoloration.
	2	-	1	Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance
	3	-	1
15 days	1	0	3	Gross Examination of the skin.
	2	-	1	Index for Erythema could not be read because of staining due to the colour of the test substance
	3	-	1	Superficial scabbing. Index for Erythema could not be read because of staining due to the colour of the test substance
Mean	1	-	3.3
	2	-	3.0
	3	-	1.7
Overall Mean		2.7

 

Note for the calculation of the mean, only the results at 24, 48 and 72 hours are used. 

PATHOLOGICAL-ANATOMICAL EVALUATION:

Skin, Left Flank: In the area of application surface raised, palpatory slight thickening of tissue.

HISTO-PATHOLOGICAL EXAMINATION:

Skin, Left Flank: Minimal Infiltration with neutrophile Granulocyte.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test substance gave a mean Edema score of 2.7 and based on the observations in this test should be classified according to EU criteria as Skin Irritant Category 2.

Executive summary:

The skin irritation potential of the test material was evaluated in this study with New Zealand White rabbits. One group of three rabbits received a volume of 0.5 ml of the test substance applied in a patch 2.5cm by 2.5cm. on the upper third of the back or the flanks. The fur was removed by clipping. The material was first heated to 90C, then cooled to body temperature for application. Following the application, each site was covered with a semiocclusive dressing. Four hours post-dose, the dressings were removed. The exposure sites were cleaned to remove as much non-absorbed test article as possible. The exposure sites were examined and scored separately for both erythema and edema at the following time periods after removal of the patch: 1h, 24h, 48h, 72h, 8d and 15d.

The evaluation of erythema was not possible, because the test substance caused red coloration of skin. The mean edema score was 2.7 and the findings were not reversible after 15 days. Pathological-Anatomical examination of 1 animal in the area of application showed surface raised, palpatory slight thickening of tissue. The Histopatholoogical examination showed minimal infiltration with neutrophil granulocyte.

Based on these observations and the edema score, the substance should be classified according to EU criteria as Skin Irritant Category 2.