disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 16, 2002 - May 24, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted July 17, 1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 31, 1992

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-050-5

EC Name:

-

IUPAC Name:

dodecasodium 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid

Details on test material:

Identity: Red Rwa 4565
Appearance: Solid, red powder
Storage: at room temperature at about 20°C

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

Recognized by the international guidelines as the recommended test system

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Scientifique des Dombes F-01400 Chatillon sur Chalaronne / France
- Age at treatment : males 10-11 weeks, females 10-12 weeks
- Weight at study initiation:
at start of acclimatization : male 1928 g, females 2035-2082 g
at start of treatment : male 2107 g, females 2139-2290 g
- Housing:lndividually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks (batch no. 1201, Provimi Kliba AG) were provided for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 116/01) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis are archived at RCC Ltd, ltingen.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, ltingen.
- 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): Air-conditioned with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours artificial fluorescent light/12 hours dark, music was played during the daytime light period

Test system

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Remarks:

Purified water

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5g test item/animal

VEHICLE
- The test item was moistened with approximately 0.1 ml purified water before application.

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

3 (animals of both sexes were used)

Details on study design:

TEST SITE
- Area of exposure: 4 cm x4 cm
- % coverage: 16% (area of patch on which the test item is applied / area of left flank that is clipped)
- Type of wrap if used: 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 (if done): the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
(indicate if minutes, hours or days): h, 24h, 48h, 72h, 7d, 10d, 14d after removal of the dressing

SCORING SYSTEM:
- Method of calculation:
The skin reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92169/EEC, July 31, 1992 approximately 1,24,48 and72 hours, as well as 7, 10 and 14 days after the removal of the dressing, gauze patch and test item. Note that the same grading is used in OECD guideline 404 (adopted: 28 July 2015). lf evident, corrosive or staining properties of the test item were described and recorded.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

erythema score

Basis:

animal: #76, #77, #78

Time point:

other: 1h , 24h, 48h, 72h, 7d, 10d, 14d

Score:

0

Max. score:

0

Reversibility:

other: not relevant in absence of effects

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

edema score

Basis:

animal: #76, #77, #78

Time point:

other: 1h, 24h, 48h, 72h, 7d, 10d, 14d

Score:

0

Max. score:

0

Reversibility:

other: not relevant in absence of effects

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

IRRITATION
Assessment of erythema was not possible in any animal t hour after treatment due to staining at the test site. The test item did not however elicit any skin reactions at the application site of any animal at any of the remaining examinations (all scores 0). The individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

CORROSION
With the exception of the staining, no irreversible alterations of the treated skin were observed nor were corrosive effects evident on the skin.

Other effects:

VIABILITY/MORTALITY/CLINICAL SIGNS
No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

COLORATION
A marked red staining was present at the test site of all animals t hour after treatment. Light red staining continued to be observed in one animal up to 72 hours after treatment and in another animal up to 10days after treatment. A light red staining was still evident in the remaining animal 14 days after treatment, the end of the observation period for all animals.

BODY WEIGHTS
The body weights of all rabbits were considered to be within the normal range of variability. Please refer to results in section "any other information on results incl. tables" hereafter.

Any other information on results incl. tables

Evolution of body weights in grams:

Body weight in grams
Animal No.	Sex	First day of acclimatization	Day of treatment	Last day of observation
76	Male	1928	2107	2557
77	Female	2082	2290	2849
78	Female	2035	2139	2614

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), with the exception of the red staining which persisted in one animal to termination of the test, RED RWa 4565 is considered to be "not irritating" to rabbit skin and is not classified according to CLP criteria.

Executive summary:

The primary skin irritation potential of RED RWa 4565 was investigated by topical semiocclusive 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, as well as 7, 10 and 14 days after removal of the dressing.

Assessment of erythema was not possible in any animal 1 hour after treatment due to staining at the test site. The application of RED RWa 4565 to the skin did not however elicit any skin reactions at the application site of any animal at any of the remaining examinations (all scores 0). The individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

The application of RED RWa 4565 resulted in marked red staining at the test site of all animals 1 hour after treatment. Light red staining continued to be observed in all animals during the observation period and was still evident in one animal 14 days after treatment, the end of the observation period for all animals. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals.