Reaction products of 4,4'-methylenebis(2,6-dimethylaniline) and 4-aminobenzenesulfonic acid with 2,4,6-trichloro-1,3,5-triazine, diazotised and subsequently coupled with N-(2-methoxyphenyl)-3-oxobutanamide

Endpoint summary

Description of key information

Skin Irritation: Ott (2006): Under the conditions of the study the test material is considered to be not irritating to rabbit skin.

Eye Irritation: Ott (2006): Under the conditions of the study the test material did not induce significant or irreversible damage to the rabbit eye.

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:

08 March 2006 to 16 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11 - 12 weeks (male); 11 - 13 weeks (females).
- Weight at study initiation (first day of treatment): 2321 g (male); 2151 - 2329 g (females).
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 % relative humidity
- Air changes (per hr): Approximately 10 - 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g (per animal) of the test material was weighed as delivered by the Sponsor
- The pH of the test material was measured before the study initiation date. A formulation of a 1 % (w/w) solution was prepared. The pH was found to be 5.

VEHICLE
- Amount(s) applied (volume or weight with unit): The test material was moistened with approximately 0.5 mL of purified water before application.

Duration of treatment / exposure:

4 h

Observation period:

72 h

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: Four days before treatment, the left flank was clipped with an electric clipper, exposing an area of approximately 100 cm² (10 cm x 10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was clipped again.
Animals with overt signs of skin injury or marked irritation which may have interfered with the interpretation of the results were not used in the test.
- Type of wrap if used: On the day of treatment, 0.5 g of the test material was placed on a surgical gauze patch (ca. 4 cm x 4 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape.

REMOVAL OF TEST SUBSTANCE
- Washing: The dressing was removed and the skin was flushed with lukewarm tap water to clean the application site.
- Time after start of exposure: The duration of treatment was 4 hours.

OBSERVATION TIME POINTS
- Viability/mortality: Daily from acclimatisation of the animals to the termination of test.
- Clinical signs: Daily from acclimatisation of the animals to the termination of test.
- Body weights: At start of acclimatisation, on the day of application and at termination of observation.
- Skin reaction: The skin reaction was assessed at approximately 1, 24, 48 and 72 hours after exposure (removal of the dressing, gauze patch and test material).
- Any lesions were described, including the degree and nature of irritation, corrosion or any other toxic effects observed, and their reversibility.

SCORING SYSTEM:
- Method of calculation: The skin reaction was assessed according to the numerical scoring system listed in the Commission Directive 2004/73/EC, 29 April 2004. If evident, corrosive or staining properties of the test material were described and recorded.
- The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. An animal is positive when the mean score is 2 or greater. The test is positive for irritation when at least 2 animals are positive for the same endpoint (erythema/eschar or oedema).
- No statistical analysis was performed.

Grading of Skin Reactions: Erythema and eschar formation
0: No erythema
1: Very slight erythema
2: Well-defined erythema
3: Moderate to severe erythema
4: Severe erythema (beef redness) or eschar formation (injuries in depth preventing erythema) reading

Grading of Skin Reactions: Oedema Formation
0: No oedema
1: Very slight oedema (barely perceptible)
2: Slight oedema (edges of area well-defined by definite raising)
3: Moderate oedema (edges raised approximately 1 mm)
4: Severe oedema (raised more than 1 mm and extending beyond the area of exposure)

NECROPSY
The animals were not sacrificed. For ethical reasons and in the interest of animal welfare, the animals were therefore re-used for the eye irritation study using the same test material.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritant / corrosive response data:

- The test material did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.
- Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.

Other effects:

- Viability, mortality and clinical signs: No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
- Colouration: Slight yellow staining produced by the test material of the treated skin was observed in all animals at the 1-hour reading and persisted in one animal until the 48-hour reading.
- Body weight: The body weights of all rabbits were considered to be within the normal range of variability.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the study the test material is considered to be not irritating to rabbit skin.

Executive summary:

The primary skin irritation potential of the test material was investigated according to OECD Test Guideline no. 404 and EU Method B.4 and in compliance with GLP.

The test material was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours after removal of the dressing.

The test material did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

The application of the test material to the skin resulted in no signs of irritation. However, slight yellow staining of the treated skin area produced by the test material was noted in all animals at the 1-hour reading and persisted in one animal until the 48-hour reading. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed.

Thus, the test material did not induce significant or irreversible damage to the skin.

Therefore, under the conditions of the study the test material is considered to be not irritating to rabbit skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

18 March 2006 to 27 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

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
- Age at study initiation: 12 – 13 weeks (male) 12 – 14 weeks (female)
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 – 23 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 10 – 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h darkness.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 g (per animal) of the test material was weighed and applied undiluted as it was delivered.
The test material was applied at 0.1 g/animal the dose specified in the test guidelines for a solid test material.
The pH of at 1 % (w/w) solution of the test material was measured for a previous study and was found to be pH 5.

Duration of treatment / exposure:

0.1 g of the test material per animal

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

Three

Details on study design:

The eyes of the animals were examined one day prior to the test material administration.
Animals with overt signs of ocular injury or irritation which may have interfered with the interpretation of the results were not used in the test.
On the day of treatment 0.1 g of the test material was placed in the conjunctival sac of the left eye of each animal 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 material. The right eye remained untreated and served as the reference control.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated eyes were not rinsed after instillation.

OBSERVATIONS
- Viability / mortality: Daily from acclimatisation of the animals to the termination of the test.
- Clinical signs: Daily from acclimatisation of the animals to the termination of the test.
- Body weights: At the start of the acclimatisation, on the day of application and at termination of observation.


SCORING SYSTEM: Grading of Ocular Lesions
Cornea
Opacity: Degree of density (area most dense taken for reading).
0: No ulceration or opacity.
1: Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible.
2: Easily discernible translucent area, details of iris slightly obscured.
3: Nacreous area, no details of visible iris, size of pupil barely discernible.
4: Opaque cornea, iris not discernible through the opacity.
Area of cornea involved:
0: Zero
1: One quarter or less
2: Greater than one quarter, but less than half
3: Greater than half, but less than three quarters
4:: Greater than three quarters, up to whole area

Iris
0: Normal
1: Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these of combination of any thereof, iris still reacting to light (sluggish reaction is positive).
2: No reaction to light, haemorrhage, gross destruction (any or all of these).

Conjunctivae
Redness (refers to most severe reading of palpebral and bulbar conjunctivae when compared with control eye).
0: Blood vessels normal.
1: Some blood vessels definitely hyperemic (injected).
2: Diffuse, crimson colour, individual vessels not easily discernible.
3: Diffuse beefy red.
Chemosis: Lids and / or nictitating membranes.
0: No swelling.
1: Any swelling above normal (including nictitating membranes).
2: Obvious swelling with partial eversion of lids.
3: Swelling with lids about half-closed.
4: Swelling with lids more than half-closed.

Additionally, ocular discharge, reddening of the sclerae and staining of conjunctivae, sclerae and cornea by the test material was assessed according to the following scheme:
Ocular discharge:
0: No discharge
1: Slight: Any amount different to normal
2: Moderate: Discharge with moistening of the lids and hair just adjacent to the lids
3: Marked: Discharge with moistening of the lids and hairs, and a considerable area around the eye (running)
Sclerae reddness
0: Normal: blood vessels normal
1: Slight reddening: Some blood vessels definitely hyperemic (injected)
2: Moderate reddening: diffuse, crimson colour, individual vessels not easily discernible
3: Marked reddening: diffuse beefy red
Staining of conjunctivae, sclerae and cornea by test material
0: Not observed
1: Slight staining
2: Marked staining

- Scleral reddening and ocular discharge were also assessed.
- The scores of each animal at the following reading times (24, 48 and 72 h) were used in calculating the respective mean values (with the exception of the sclerae) for each type of lesion.

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic lamp (RothAG, CH-4153, Reinach, Switzerland). The eyes of each animal were examined approximately 1, 24, 48 and 72 h after administration.

PATHOLOGY
- No necropsy was performed on animals treated at termination of observation.
- All rabbits were sacrificed by intravenous injection of Vetanarcol into the ear vein at a dose of at least 1 mL/kg body weight (equivalent to 162 mg sodium pentobarbitone / kg bodyweight) and discarded.

STATISTICAL ANALYSIS
No statistical analysis was performed.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Reddening

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Reddening

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Reddening

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The mean score was calculated across 3 scoring times (24, 48 and 72 h after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately.
No abnormal findings were observed in the iris of any animal at any of the measurement intervals.
Very slight corneal opacity affecting the whole area at the 1 h reading and affecting one quarter (or less) but not zero was noted in one animal.
Moderate reddening of the conjunctivae was noted in all animals at the 1-h reading and persisted as slight reddening until the 24 h reading in two animals, and until the 48 h reading in one animal.
Slight swelling (chemosis) of the conjunctivae to obvious swelling with partial eversion of the lids was observed in all animals at the 1 h reading.
Slight to moderate reddening of the sclerae was present in all animals at the 1 h reading and persisted as slight reddening in two animals at the 24 h reading.
Slight ocular discharge was noted in all animals at the 1 h reading.
No abnormal findings were observed in the treated eye of any animal 72 h after treatment, the end of the observation period for all animals.
No corrosion of the cornea was observed at any of the reading times.

Other effects:

- Lesions and clinical observations: No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
Other observations:
- Colouration: Yellow remnants of the test material were observed in the eye or conjunctival sac of all animals one hour after treatment.
- Bodyweights: One animal showed a slight loss of body weight (0.9 %) at the end of the observation period. The bodyweights of the remaining rabbits were considered to be within the normal range of variability.

Eye Irritation Scores – Individual Values

Animal No.	Sex	Evaluation interval*	Corneal opacity	Area of corneal opacity	Iris	Conjunctivae		Sclera
						Redness	Chemosis
1	M	1 h	1	4	0	2	2	2
2	F		0	0	0	2	1	1
3	F		0	0	0	2	1	1
1	M	24 h	1	1	0	1	0	1
2	F		0	0	0	1	0	0
3	F		0	0	0	1	0	1
1	M	48 h	0	0	0	1	0	0
2	F		0	0	0	0	0	0
3	F		0	0	0	0	0	0
1	M	72 h	0	0	0	0	0	0
2	F		0	0	0	0	0	0
3	F		0	0	0	0	0	0

* Examinations were performed at the specified times after installation of the test material.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the study the test material did not induce significant or irreversible damage to the rabbit eye.

Executive summary:

The primary irritation potential of the test material was investigated according to OECD Test Guideline 405 and EU Method B.5 and in compliance with GLP.

The test material was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 h after test material instillation.

The mean score was calculated across three scoring times (24, 48 and 72 h after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual means scores for iris were 0.0 for all three animals. The individual means scores for corneal opacity were 0.33, 0.0 and 0.0 respectively. The individual means scores for the conjunctivae were 0.67, 0.33 and 0.33 for reddening and 0.0 for chemosis for all three animals.

The instillation of the test material into the eye resulted in mild, early-onset and transient ocular changes such as corneal opacity, reddening of the sclerae, discharge and chemosis. These effects were reversible and were no longer evident 72 h after treatment, the end of the observation period for all animals. No abnormal findings were observed in the iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Yellow remnants of the test material were observed in the eye or conjunctival sac of all animals on hour after treatment. No clinical signs were observed.

Under the conditions of the study the test material did not induce significant or irreversible damage to the rabbit eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation: Ott (2006)

The primary skin irritation potential of the test material was investigated according to OECD Test Guideline no. 404 and EU Method B.4 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours after removal of the dressing.

The test material did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

The application of the test material to the skin resulted in no signs of irritation. However, slight yellow staining of the treated skin area produced by the test material was noted in all animals at the 1-hour reading and persisted in one animal until the 48-hour reading. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed.

Thus, the test material did not induce significant or irreversible damage to the skin.

Therefore, under the conditions of the study the test material is considered to be not irritating to rabbit skin.

Eye Irritation: Ott (2006)

The primary irritation potential of the test material was investigated according to OECD Test Guideline 405 and EU Method B.5 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 h after test material instillation.

The mean score was calculated across three scoring times (24, 48 and 72 h after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual means scores for iris were 0.0 for all three animals. The individual means scores for corneal opacity were 0.33, 0.0 and 0.0 respectively. The individual means scores for the conjunctivae were 0.67, 0.33 and 0.33 for reddening and 0.0 for chemosis for all three animals.

The instillation of the test material into the eye resulted in mild, early-onset and transient ocular changes such as corneal opacity, reddening of the sclerae, discharge and chemosis. These effects were reversible and were no longer evident 72 h after treatment, the end of the observation period for all animals. No abnormal findings were observed in the iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Yellow remnants of the test material were observed in the eye or conjunctival sac of all animals on hour after treatment. No clinical signs were observed.

Under the conditions of the study the test material did not induce significant or irreversible damage to the rabbit eye.

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 skin or eye irritation or corrosion.