N-(2-ethylhexyl)-1-[[2-methyl-4-[(2-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:

29.10.2012 - 9.11.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

260-124-8

EC Name:

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

Cas Number:

56358-09-9

Molecular formula:

C32H37N5

IUPAC Name:

N-(2-ethylhexyl)-1-({2-methyl-4-[(2-methylphenyl)diazenyl]phenyl}diazenyl)-1,2-dihydronaphthalen-2-amine

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Solvent Red 19E
- Physical state: dark viscous liquid/borderline waxy solid
- Analytical purity: 90% (w/w)
- Impurities (identity and concentrations): Solvent Red24 (CAS 85-83-6) 2% (w/w)
- Lot/batch No.: S2409
- Expiration date of the lot/batch: unlisted
- Storage condition of test material: in tightly closed container in dark and well ventilated place at room temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.8-4.5 kg
- Housing: Individually in cages without bedding
- Diet (e.g. ad libitum): Pelleted standard diet for rabbits and guinea-pigs ad libitum
- Water (e.g. ad libitum): Drinking tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Relative humidity (%): 30-70 %
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

STUDY TIME SCHEDULE
Animal supply: 17.10.2012
Experimental part of study: 29.10.2012 – 09.11.2012

Test system

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mg

Duration of treatment / exposure:

4 h

Observation period:

1, 24, 48, 72 hours

Number of animals:

Initial test: 1 female
Confirmatory test: 2 females

Details on study design:

TEST SITE
- Area of exposure: 6 x 6 cm
- Type of wrap if used: Gauze patch, foil and cellulose cotton and held in place with non-irritating tape - Spofaplast

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period the patch was removed and the remaining sample was washed with olive oil.

SCORING SYSTEM: According to the grading system given in Method B.4 Acute toxicity: Dermal Irritation/Corrosion, Council Regulation (EC) No.440/2008

Results and discussion

In vivo

Results

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: Due to colouration of test substance the evaluation of erythema was not carried out.

Irritant / corrosive response data:

Initial Test (One Animal)
The test substance was applied to the skin of one rabbit approximately 24 hours after fur removal. The first patch was removed after three minutes. Due to colouration of the test substance, the evaluation of erythema was not carried out, because the test substance caused red colouration of the skin. But no serious skin reactions were observed, so the second patch was applied for one hour, after which no serious skin reactions were observed. The third patch was applied for 4 hours after which the response was graded. Because no corrosive effect was observed after a 4-hour exposure, the response was verified in confirmatory test, using two additional animals, each with one patch for 4 hours.
Due to colouration of the test substance the evaluation of erythema was not carried out because the test substance caused red colouration of the skin, no oedema was observed after 4 hour exposure in the rabbit. At 1, 24, 48 and 7 hours, after exposure, no sign of oedema were recorded and no evidence of a corrosive effect was observed on the skin of the rabbit. Clinical observation was carried out for 7 days. The rabbit was killed 6 days after exposure.
The skin was removed for histopathological examination after humane sacrifice of the animal, because the evaluation of skin reactions could not be performed.

Confirmatory Test
Skin reaction was evaluated after patch removal after 1, 24, 48 and 72 hours. No corrosive skin reaction was evaluated during all observation periods but evaluation of erythema was not performed because the test substance caused red colouration of skin. The rabbits were killed 4 days after exposure.

Other effects:

Results of Clinical Observations
Clinical observations were carried out throughout the whole study until the sacrifice of animals. There was no evidence of corrosive effect on the skin. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Histopathological examination of skin was performed.

Histopathological examination
Because the evaluation of erythema could not be carried out, the histopathological examination was performed.
Histopathological examination of skin as performed on all rabbits. The following samples of skin were collected at necropsy:
Rabbit no. 10: Skin area without exposure, skin rea with exposure for 3 mins, skin area with exposure time 1 hour and skin area with exposure time 4 hours.
Rabbit no. 11: Skin area without exposure, skin area with exposure time 4 hours.
Rabbit no. 12: Skin area without exposure, skin area with exposure time 4 hours.
Skin area without exposure was taken from the rear part of the trunk. Skin specimens were fixed in 4 % buffer formaldehyde and then processed by routine paraffin technique and stained by haematoxylin-eosin.
Diffuse mixed infiltration (mononuclear cells and granulocytes) in dermis was observed in all rabbits regardless of the time of exposure. Small haemorrhage in sub-epidermis occurred in two rabbits (rabbit 10 after 3-minutes exposure and rabbit 12 after 4-hours exposure). Sub-epidermal oedema was also detected in rabbit 10 after 1 hour exposure diffuse oedema and hyperaemia were detected after 4-hours exposure.

Any other information on results incl. tables

Pathological Examination of Skin: Rabbit No 10.

Sample	Histopathological Changes
Control skin	Without pathological changes
Skin with exposure time 3 mins	Diffuse mix of infiltration (mononuclear cells and granulocytes) in dermis. Small haemorrhage in sub-epidermis. Necrosis undetected.
Skin with exposure time 1 hour	Diffuse mixed infiltrations (mainly mononuclear cells) in dermis. Sub-epidermal oedema. Necrosis undetected.
Skin with exposure time 4 hours	Diffuse mixed infiltration (mononuclear cells and granulocytes) in dermis. Diffuse oedema and hyperaemia mainly in sub-epidermis. Necrosis undetected.

 

Pathological Examination of Skin: Rabbit No 11.

Sample	Histopathological Changes
Control skin	Without pathological changes
Skin with exposure time of 4 hours	Diffuse mixed infiltration (mononuclear cells and granulocytes) in dermis. Necrosis undetected.

 

 

Pathological Examination of Skin: Rabbit No. 12

Sample	Histopathological Changes
Control skin	Without pathological changes
Skin with exposure time 4 hours	Diffuse mixed infiltration (mononuclear cells and granulocytes) in dermis. Small focal haemorrhage in sub-epidermis. Necrosis undetected.

 

Applicant's summary and conclusion

Interpretation of results:

irritating

Conclusions:

The test material Solvent Red 19E did not cause corrosion of skin, but during the histopathological examination it was found that the test substance caused dermal tissues changes that indicate irritation of skin.

Executive summary:

The test material was assessed for acute dermal irritation/corrosion according to Method B.4 - Acute Toxicity: Dermal Irritation/Corrosion, Council Regulation (EC) No.440/2008, and OECD Test Guideline 404.

Three rabbits were exposed to 0.5 mg of test substance, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were evaluated after patch removal and observations were made at 1, 24, 48 and 72 hours after exposure.

At first the test substance was applied on the skin of one for 3 minutes, 1 hour and 4 hours. No signs of oedema or corrosion were seen, so confirmatory tests with two others rabbits were performed with a 4-hour exposition period.

The evaluation of erythema was not possible, because the test substance caused red coloration of skin. But no oedema was observed after 4-hour exposure in all rabbits. At 1, 24, 48 and 72 hours after exposure no sign of oedema were recorded and no evidence of a corrosive effect was observed on the skin of any of the three rabbits tested.

Because the evaluation of erythema was not carried out, the histopathological examination of skin was performed on all rabbits. The animals were humanely killed and histological preparations were prepared from exposed and control skin. Diffuse mixed infiltration (mononuclear cells and granulocytes) in dermis was observed in all rabbits regardless the time of exposure. Small haemorrhage in sub-epidermis occurred in two rabbits. Sub-epidermal oedema, diffuse oedema and hyperaemia were also detected in one rabbit.

The test material did not cause corrosion of skin. During the histopathological examination it was found that the test substance caused dermal tissues changes that indicate irritation of skin, however this cannot be quantified due to the nature of the test material and its colouring of the test sites.