Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Endpoint summary

Description of key information

Skin: The test item was considered to be non-irritating based on a primary irritation index of zero (OECD 404 and EU Method B.4)

Eye: Irritation of the conjunctivae which consisted of redness in one animal which resolved within 24 hours and redness and discharge in the other two animals which resolved within 48 hours. No iridial or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage (OECD 405 and EU Method B.5).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 October 2008 - 08 November 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study; well documented study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

albino; SPF-quality

Details on test animals or test system and environmental conditions:

ANIMALS
- Source: Harlan France SARL, Gannat, France
- Animals used within the study were at least 6 weeks old and body weights were at least 1.0 kg
- Identification: Earmark
- Health inspection: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health. Special attention was paid to the skin to be treated, which was intact and free from abnormalities.

ANIMAL HUSBANDRY
- Animals were housed in a controlled environment in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0 °C (acutal range 22.2 to 23.2 °C), a relative humidity of 30 to 70 % (actual range 32 to 64 %) and 12 hours artificial fluorescent light and 12 hours darkness per day.

ACCOMODATION
- Animals were individually housed in labelled cages with perforated floors (Ebeco, Germany) dimensions 67 x 62 x 55 cm and shelters (Ebeco, Germany) dimensions 40 x 32 x 23 cm.
- Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

DIET
- Pelleted diet for rabbits (Global Diet 2030 (irradiated) Mucedola srl, Sellimo Milanese, Italy, supplied by Harlan Netherlands B.V., Horst, The Netherlands approximately 100 g per day plus hay (TecniLab-BMI BV, Someren, The Netherlands) provided at least three times per week.

WATER
- Free access to tap water.

ANALYSIS
- Results of analysis for each batch of diet (nutrients and contaminants), hay and water were assessed and did not reveal any findings that were considered to have affected the integrity of the study.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

other: Adjacent areas of the untreated skin of each animal served as controls

Amount / concentration applied:

TEST MATERIAL
- Test substance was ground to a powder using a pestle and mortar prior to weighing and the test substance was moistened with water (Elix, Millipore S.A.S, Molsheim, France) immediately before application to ensure close contact with the animal's skin.
- Amount(s) applied (volume or weight with unit): 0.5 grams of test substance

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48 and 72 hours

Number of animals:

3 males

Details on study design:

STUDY DESIGN
- An in vitro study was considered but a negative result was anticipated and this would need to have been confirmed by an in vivo study.
- The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel).
- The two other animals were treated in a similar manner one wek later after considering the degree of skin irritation observed in the first animal.
- No histopathology was performed.

TREATMENT
- Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers exposing an area of approximately 10 x 15 cm (150 cm2).
- To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations.
- The test substance (0.5 g) was moistened with 0.8 mL of the vehicle and applied to the skin of one flank, using a metalline patch of 2 x3 cm.
- The patch was mounted on a Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage.
- Four hours after the application, the dressing was removed and the skin cleansed of residual test substance using tap water and watery ethanol (50% v/v).

OBSERVATIONS
- Mortality/viability: Twice daily
- Toxicity: At least once daily
- Body weight: Day of treatment (prior to application) and at termination
- Irritation: The eyes of each animal were examined at approximately 1, 24, 48 and 72 hours after removal of the dressings. Irritation scores according to the attached scheme were recorded together with a description of other (local effects).

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 966

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 26

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 28

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 966

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 26

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 28

Irritant / corrosive response data:

- Individual skin irritation scores are presented in Table 1 (attached).
- No skin irritation was caused by exposure to the test item for four hours.
- There was no evidence of a corrosive effect on the skin.

Other effects:

- Grey/brown staining of the treated skin by the test substance was observed on Day 1 which did not hamper the scoring of the skin reactions. No remnants of the test substance were present on the skin.
- No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

not irritating

Conclusions:

The test item was considered to be non-irritating based on a primary irritation index of zero.

Executive summary:

GUIDELINE

The study was conducted in accordance with OECD No 404 (2002) "Acute Dermal Irritation/Corrosion", EU No Method B.4 (2008) "Acute Toxicity: Dermal Irritation/Corrosion", EPA OPPTS 870.2500 (1998) "Acute Dermal Irritation" and JMAFF guidelines (2000) including the most recent revisions.

METHODS

Three rabbits were exposed to test item (0.5 g) moistened with water by application onto clipped skin for 4 hours using a semi-occlusive dressing. Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers exposing an area of approximately 10 x 15 cm (150 cm2). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations. The test substance (0.5 g) was moistened with 0.8 mL of the vehicle and applied to the skin of one flank, using a metalline patch of 2 x3 cm. The patch was mounted on a Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage. Four hours after the application, the dressing was removed and the skin cleansed of residual test substance using tap water and watery ethanol (50% v/v).

RESULTS

No skin irritation was caused by exposure to the test item for four hours. Grey/brown staining of the treated skin by the test substance was observed on Day 1, which did not hamper the scoring of skin reactions. No remnants of the test substance were present on the skin.

CONCLUSION

The test item was considered to be non-irritating based on a primary irritation index of zero.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2008 - 13 November 2008

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan France SARL, Gannat, France.
- Conditions: Controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 +/- 3C, a relative humididty of 30-70% and 12 hours artificial fluorescent light and 12 hours darkness per day.
- Housing: Individually in labelled cages with perforated floors (Ebeco, Germany) dimensions 67 x 62 x 55 cm and shelters (Ebeco, Germany) dimensions 40 x 32 x 23 cm.
- Acclimatisation: At least five days before the start of treatment under laboratory conditions.
- Diet: Pelleted diet for rabbits (K-H from SSNIFF Spezialdiatenj GmbH, Soest, Germany (approximately 100 g per day) and hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least three times a week.
- Water: Free access to tap water.
- Results of analysis for each batch of diet (nutrients and contaminants), hay and water were assessed and did not reveal any findings that were considered to have affected the integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

other: The other eye remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): Undiluted
- Amount(s) applied (volume or weight with unit): 62.1 mg (range 61.8-62.2 mg) of the test substance (a volume of approximately 0.1 mL)

Duration of treatment / exposure:

72 hours in one animal (sentinel) and 24 hours in the other two animals.

Observation period (in vivo):

approximately 1, 24, 48, and 72 hours after instillation

Number of animals or in vitro replicates:

3 Males

Details on study design:

STUDY DESIGN
- An in vitro study was considered but a negative result was anticipated and this would need to have been confirmed by an in vivo study.
- The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel).
- The two other animals were treated in a similar manner one wek later after considering the degree of eye irritation observed in the first animal.

TREATMENT
- Animals were treated by instillation of 62.1 to 62.2 mg test substance (0.1 mL) into the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball.
- The lids were then gently held together for about one second to prevent loss of the test substance.
- The other eye remained untreated and served as the reference control.
- Immediately after the 24 -hour observation, a solution of 2 % fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.
- Immediately after fluorescein examination on Day 2, the treated eye of two animals was rinsed with tepid tap water (approximately 50 mL) to remove residual test substance using a velocity of flow which did not affect the eye.
- For reference control the other eye was also rinsed.
- After the final observation, the animals were sacrificed by intravenous injection of phenobarbital (Euthesate; Ceva Sante Animale BV, Naaldwijk, The Netherlands).

OBSERVATIONS
- Mortality/viability: Twice daily
- Toxicity: At least once daily
- Body weight: Day of treatment (prior to instillation) and at termination
- Irritation: The eyes of each animal were examined at approximately 1, 24, 48 and 72 hours after instillation of the test substance. Irritation scores according to the attached scheme were recorded together with a description of other (local effects).

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 955

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: Animal 955

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: not applicable

Remarks on result:

other: Animal 955

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 955

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 3

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: Animal 3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Animal 3

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 4

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: Animal 4

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Animal 4

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Animal 4

Irritant / corrosive response data:

- Individual eye irritation scores are presented in Table 1 (attached).
- Instillation of approximately 62 mg of test item (0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjuntivae (redness in one animal which resolved within 24 hours and redness and discharge in the other two animals which resolved within 48 hours).
- No iridial irritation or corneal opacity was observed and treatment of the eyes with 2 % fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.
- There was no evidence of ocular corrosion.

- No staining of (peri) ocular tissues by the test substance was observed. Remnants of the test substance were present in the eye and on the outside of the eyelids in all animals on Day 1. On Day 2, test substance remnants were still present in the eye in two animls and on the outside of the eyelids in one animal.

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

Interpretation of results:

not irritating

Conclusions:

Instillation of approximately 62 mg of test item (a volume of approximately 0.1 mL) into the eye of each of three rabbits resulted in irritation of the conjunctivae which consisted of redness in one animal which resolved within 24 hours and redness and discharge in the other two animals which resolved within 48 hours. No iridial or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Based on these results the test item does not have to be classified for eye irritation according to EC criteria (Council Directive 67/548/EEC) or the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007).

Executive summary:

GUIDELINE

The study was conducted in accordance with OECD No 405 (2002) "Acute Eye Irritation/Corrosion", EU No Method B.5 (2008) "Acute Toxicity: Eye Irritation/Corrosion", EPA OPPTS 870.2400 (1998) "Acute Eye Irritation" and JMAFF guidelines (2000) including the most recent revisions.

METHODS

Animals were treated by instillation of 62.1 to 62.2 mg test substance (0.1 mL) into the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control. Immediately after the 24 -hour observation, a solution of 2 % fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Immediately after fluorescein examination on Day 2, the treated eye of two animals was rinsed with tepid tap water (approximately 50 mL) to remove residual test substance using a velocity of flow which did not affect the eye. For reference control the other eye was also rinsed. After the final observation, the animals were sacrificed by intravenous injection of phenobarbital.

RESULTS

Instillation of approximately 62 mg of test item (a volume of approximately 0.1 mL) into the eye of each of three rabbits resulted in irritation of the conjunctivae which consisted of redness in one animal which resolved within 24 hours and redness and discharge in the other two animals which resolved within 48 hours. No iridial or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

CONCLUSION

Based on study results the test item does not have to be classified for eye irritation according to EC criteria (Council Directive 67/548/EEC) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

A key study was conducted in accordance with OECD No 404 (2002) "Acute Dermal Irritation/Corrosion", EU No Method B.4 (2008) "Acute Toxicity: Dermal Irritation/Corrosion", EPA OPPTS 870.2500 (1998) "Acute Dermal Irritation" and JMAFF guidelines (2000) including the most recent revisions.

Three rabbits were exposed to test item (0.5 g) moistened with water by application onto clipped skin for 4 hours using a semi-occlusive dressing. Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers exposing an area of approximately 10 x 15 cm (150 cm2). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations. The test substance (0.5 g) was moistened with 0.8 mL of the vehicle and applied to the skin of one flank, using a metalline patch of 2 x3 cm. The patch was mounted on a Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage. Four hours after the application, the dressing was removed and the skin cleansed of residual test substance using tap water and watery ethanol (50% v/v).

No skin irritation was caused by exposure to the test item for four hours. Grey/brown staining of the treated skin by the test substance was observed on Day 1, which did not hamper the scoring of skin reactions. No remnants of the test substance were present on the skin.

The test item was considered to be non-irritating based on a primary irritation index of zero.

Eye

A key study was conducted in accordance with OECD No 405 (2002) "Acute Eye Irritation/Corrosion", EU No Method B.5 (2008) "Acute Toxicity: Eye Irritation/Corrosion", EPA OPPTS 870.2400 (1998) "Acute Eye Irritation" and JMAFF guidelines (2000) including the most recent revisions.

Animals were treated by instillation of 62.1 to 62.2 mg test substance (0.1 mL) into the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control. Immediately after the 24 -hour observation, a solution of 2 % fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Immediately after fluorescein examination on Day 2, the treated eye of two animals was rinsed with tepid tap water (approximately 50 mL) to remove residual test substance using a velocity of flow which did not affect the eye. For reference control the other eye was also rinsed. After the final observation, the animals were sacrificed by intravenous injection of phenobarbital.

Instillation of approximately 62 mg of test item (a volume of approximately 0.1 mL) into the eye of each of three rabbits resulted in irritation of the conjunctivae which consisted of redness in one animal which resolved within 24 hours and redness and discharge in the other two animals which resolved within 48 hours. No iridial or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Based on study results the test item does not have to be classified for eye irritation according to EC criteria (Council Directive 67/548/EEC) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007).

Justification for classification or non-classification

Skin

The substance does not meet the criteria for classification as an eye irritant under the terms of Regulation No. 1272/2008.

Eye

The substance does not meet the criteria for classification as an eye irritant under the terms of Regulation No. 1272/2008.