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

Endpoint summary

Description of key information

In vitro skin corrosion test (EU B.40, GLP compliant) - negative result

In vitro skin irritation test (EU B.46, GLP compliant) - negative result

In vivo skin irritation/corrosion test (EU B.4, GLP compliant) - positive result

 

In vitro BCOP test (EU B.47, GLP compliant) - negative result

In vivo eye irritation/corrosion test (EU B.5, GLP compliant) - negative result

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

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

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

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.

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.

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5.11.2012 - 12.11.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 3.4 - 4 kg
- Housing: Individually in metallic cages
- Diet (e.g. ad libitum): Standard pelleted diet for rabbits and guinea pigs ad libitum
- Water (e.g. ad libitum): Drinking tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Relative humidity (%): 30-70 %
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

STUDY TIME SCHEDULE
Animal supply: 17.10. 2012
Experimental part of study: 5.11.2012 – 12. 11. 2012

Vehicle:

other: olive oil

Remarks:

10 % w/v solution

Controls:

other: The second eye was treated with olive oil and served as a control.

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 10 % (w/v) solution in olive oil. This was the maximum technically practicable.
- The dose of 0.1 mL of test substance solution was applied to the eye of the animal by syringe.

VEHICLE
- Lot/batch no. (if required): 5211201

Duration of treatment / exposure:

72 h

Observation period (in vivo):

1, 24, 48 and 72 hrs

Number of animals or in vitro replicates:

Initial test: 1 female
Confirmatory test: 2 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM: According to the grading system given in Method B.5 Acute Toxicity: Eye Irritation/Corrosion

TOOL USED TO ASSESS SCORE: After recording the observations at 72 hours, the eyes of rabbit were examined with the aid of fluorescein and the ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

The control eyes in all rabbits were without ocular lesions during the whole study.

Other effects:

No symptoms of systemic toxicity were observed in the animals during clinical observations in the test period and no mortality occurred.

Interpretation of results:

not irritating

Conclusions:

The test material is not irritating to the eyes.

Executive summary:

The test material was assessed for eye irritation/corrosion according to the Method B.5 Acute Toxicity: Eye Irritation/Corrosion. Council Regulation (EC) No.440/2008, and OECD Test guideline 405.

The test was performed initially using one animal. Because no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals.

No pathological changes of eyes of all rabbits were observed during whole study. During the observation period (at 1, 24, 48 and 72 hours after application) no irritating effects of eye was observed in all animals.

No clinical signs of systemic intoxication were detected.

Evaluation of results after single application demonstrated that the test substance is not irritating for eye of rabbit.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN

IN VITRO SKIN CORROSION

Test material was assessed for skin corrosion according to Method B. 40. Skin corrosion (in vitro), Council Regulation (EC) No.440/2008 and OECD Test Guideline 431.

The test substance was placed atop the previously moistened tissue with the aid of application pin. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three each per test substance, positive control and negative control.

After rinsing, tissues were incubated with MTT for three hours and extracted overnight subsequently at room temperature without shaking. OD570 of isopropylalcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, average viability of tissues treated by the test substance Solvent Red 19E was 78.6 % of negative control average value after 3 min treatment and 118.3 % after 60 min treatment.

In the experiment arrangement given above, the test substance Solvent Red 19E was non-corrosive in EpiDermTM model.

IN VITRO SKIN IRRITATION

Test substance was assessed for skin irritation according to EU Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDerm™ Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT, and OECD Test Guideline 439.

After pre-incubation of tissues, the test substance was pressed atop the tissue with the aid of application pin. Length of exposition was 60 minutes. Twelve tissues were used for the experiment, six per test substance and three each for positive control (PC) and three for negative control (NC).

After rinsing, tissues were post incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, average viability of tissues treated by the test substance was 111. 0% of negative control average value (i.e. viability was greater than limit value 50 %).

The effect of test substance was negative in EpiDerm™ model (the tissue was not damaged).

According to the classification criteria, the test substance is considered to have no category in regard to skin irritation.

IN VIVO SKIN IRRITATION/CORROSION

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.

EYE

IN VITRO BCOP TEST

The test material was assessed for ocular corrosivity or severe irritancy according to the Method B.47 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council Regulation (EC) No.1152/2010, and OECD Test Guideline 437.

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: Test substance treatment group, positive control group and negative control group. Three corneas per group were used.

An open-chamber method was used, because the test substance was viscous liquid/borderline waxy solid. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Solvent Red 19E was 1.37. This is less than the limit value of IVIS (55.1), therefore the test substance was not identified as a corrosive or severe irritant.

IN VIVO EYE IRRITATION/CORROSION

The test material was assessed for eye irritation/corrosion according to the Method B.5 Acute Toxicity: Eye Irritation/Corrosion. Council Regulation (EC) No.440/2008, and OECD Test guideline 405.

The test was performed initially using one animal. Because no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals.

No pathological changes of eyes of all rabbits were observed during whole study. During the observation period (at 1, 24, 48 and 72 hours after application) no irritating effects of eye was observed in all animals.

No clinical signs of systemic intoxication were detected.

Evaluation of results after single application demonstrated that the test substance is not irritating for eye of rabbit.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to eye irritation.

 

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification with respect to skin irritation as Category 2 (H315; Causes skin irritation).