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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 4-{4-[4-(4-Amino-3,5-dimethyl-benzyl)-2,6-dimethyl-phenylamino]-6-chloro-[1,3,5]triazin-2-ylamino}-benzenesulfonic acid, sodium nitrite, hydrochloric acid and acetoacet-o-anisidin

Cas Number:

1165939-52-5

IUPAC Name:

Reaction product of 4-{4-[4-(4-Amino-3,5-dimethyl-benzyl)-2,6-dimethyl-phenylamino]-6-chloro-[1,3,5]triazin-2-ylamino}-benzenesulfonic acid, sodium nitrite, hydrochloric acid and acetoacet-o-anisidin

Test material form:

solid: particulate/powder

Remarks:

Yellow solid

Details on test material:

- Expiry date: 20 Dec 2007
- Storage conditions: At room temperature (ca 20°C)

Test animals

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.

Test system

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.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Results 2

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Results 3

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Results 4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Results 5

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Results 6

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.

Applicant's summary and conclusion

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.