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

Endpoint summary

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:

April 16, 2002 - May 24, 2002

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:

adopted July 17, 1992

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)

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

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.

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

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.

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

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.

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:

May 14, 2002 - July 16, 2002

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:

adopted in February 24, 1987

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 31, 1992

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elevage Scientifique des Dombes F-01400 Chatillon sur Chalaronne / France
- Age at study initiation (at start of treatment): 11 weeks for male (animal #40) and 11 and 15 weeks for females (animal #41 and #42)
- Weight at study initiation (first day of acclimatization): 2150 g (male #40), 2110 g (female #41) and 2013 g (female #42)
- Housing: lndividually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks (batch no. 0203, Provimi Kliba AG) were provided for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad tibitum (batch no.25102) 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
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other:

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1g/eye
- Concentration (if solution): undiluted

Duration of treatment / exposure:

21 days as the treated eyes were not rinsed after instillation

Observation period (in vivo):

21 days after application

Number of animals or in vitro replicates:

3 (male #40, females #41 & #42)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none
- Time after start of exposure: not relevant

SCORING SYSTEM: The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92l69lEEC, July31, 1992 at approximately 1, 24, 48 and 72 hours, as well as 7, 10,14,17 and 21 days after application.

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, CH-41 53 Reinach/Switzerland).

Irritation parameter:

cornea opacity score

Basis:

animal: #40, #41 & #42

Time point:

other: 1h, 24h, 48h, 72h, 7d, 10d, 14d, 17d, 21d

Score:

0

Max. score:

0

Reversibility:

other: not relevant as scores remain nul

Irritation parameter:

other: Area of Corneal Opacity

Basis:

animal: #40, #41 & #42

Time point:

other: 1h, 24h, 48h, 72h, 7d, 10d, 14d, 17d, 21d

Score:

0

Max. score:

0

Reversibility:

other: Not relevant as scores remain nul

Irritation parameter:

iris score

Basis:

animal: #40, #41 & #42

Time point:

other: 1h, 24h, 48h, 72h, 7d, 10d, 14d, 17d, 21d

Score:

0

Max. score:

0

Reversibility:

other: not relevant as scores remain nul

Irritation parameter:

conjunctivae score

Basis:

animal: #40

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal: #40

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal: #41

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal: #42

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal: #42

Time point:

other: 48/72h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: #42

Time point:

24/48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

IRRITATION
Application of the test item to healthy rabbit conjunctivae resulted in a primary eye irritation score of 1.11.
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately.
The individual mean score for corneal opacity and iris was 0.00 for all three animals.
The individual mean scores for the conjunctivae were 0.33, 0.33 and 1.33 for reddening and 0.33, 0.33 and 0.67 for chemosis, respectively.

No abnormal findings were observed in the cornea or iris of any animal at any of the measuring intervals.
Assessment of reddening of the conjunctivae was not possible in any animal at the 1-hour examination due to the presence of staining in the eye and/or conjunctival sac. Slight to moderate reddening was, however, noted in all animals 24 hours after treatment and slight reddening continued to be observed in one animal up to the 72 hour examination.. Obvious swelling with partial eversion of lids or swelling with lids about half closed was apparent in all animals 1 hour after treatment. Slight swelling was observed in all animals at the 24hour examination and again in one animal at the 48 hour examination.
Full assessment of the sclerae was also prevented at the 1 hour examination due to ocular staining. Slight reddening of the sclerae was however subsequently observed in all animals during the observation period.
Discharge with moistening of the lids and hairs just adjacent to the lids was evident in all animals 1 hour after treatment. lncreased ocular discharge was similarly observed in one animal at the 24 hour examination.

CORROSION
No corrosion of the cornea was observed at any of the reading times.

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.

COLORATION
Marked red staining was present in the treated eye of all animals 1 hour after instillation of the test item. Slight staining continued to be observed in all animals during the entire observation period and was still evident at termination,21 days after treatment.
Red remnants of the test item were observed in the eye or conjunctival sac of 1 animal 1 hour after treatment.

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

Eye irritation scores - individual values

Animal number	Sex	Evaluation Interval*	Corneal Opacity	Area of Corneal Opacity	Iris	Conjunctivae		Cumulative		Sclera
						Redness	Chemosis	Score	Mean
40	M	1 hour	0	0	0	n.a.	2	2.00	2.33	n.a.
41	F		0	0	0	n.a.	2	3.00		n.a.
42	F		0	0	0	n.a.	3	3.00		n.a.
40	M	24 hours	0	0	0	1	1	2.00	2.33	1
41	F		0	0	0	1	1	2.00		1
42	F		0	0	0	2	1	3.00		1
40	M	48 hours	0	0	0	0	0	0.00	0.67	0
41	F		0	0	0	0	0	0.00		1
42	F		0	0	0	1	1	2.00		1
40	M	72 hours	0	0	0	0	0	0.00	0.33	0
41	F		0	0	0	0	0	0.00		0
42	F		0	0	0	1	0	1.00		0

*Examinations were performed at the specified times after instillation of the test item

n.a.= not assessable due to staining produced by the test item

Note: Observations continued for 21 days due to staining present in the eye

 

At 7, 10, 14, 17, 21 days after treatment, the eye reactions for the three assessed animals consisted of grade 0.00 for corneal opacity, area of corneal opacity, iris and conjunctivae (redness and chemosis).

Evolution of body weights in grams

Body weight in grams
Animal No.	Sex	First day of acclimatization	Day of treatment	Last day of observation
40	Male	2150	2393	2922
41	Female	2110	2219	3003
42	Female	2013	2339	2935

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on criteria defined in Regulation (EC) No 1272/2008, RED RWa 4565 is classified as Serious eye damage (category 1). Eye irritation test shows persistence of red staining at the end of the 21 days recovery period which is according to the CLP guideline a reason for Serious eye damage Cat 1 (discoloration of the cornea by a dye substance).

Executive summary:

The primary eye irritation potential of RED RWa 4565 was investigated by instillation of 0.1 g into one eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7, 10, 14, 17 and 21 days after test item application. The scores of each animal at the following reading times (24, 48 and72 hours) were used in calculating the respective mean values for each type of lesion.

The primary eye irritation score was calculated by totaling the mean cumulative scores at 24, 48 and 72 hours and then dividing the resulting total by the number of data points. The primary eye irritation score was 1.11 (max. 13).

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean score for corneal opacity and iris was 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.33, 0.33 and 1.33 for reddening and 0.33, 0.33 and 0.67 for chemosis, respectively.

The instillation of RED RWa 4565 into the eye resulted in mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 7 days after treatment. Where evaluation was possible, no abnormal findings were observed in the cornea or iris of any animal. No corrosion was observed at any of the measuring intervals. A marked red staining was, however, present in the treated eye of all animals t hour after instillation of the test item. Slight staining continued to be observed in all animals during the entire observation period and was still evident at termination, 21 days after treatment.

Based upon the referred classification (Commission Directive 2001/59/EC of August 06, 2001), due to the irreversible red staining present in the treated eyes of all animals, RED RWa 4565 is considered to be "R41-Risk of serious damage to eyes".

Based on criteria defined in Regulation (EC) No 1272/2008, RED RWa 4565 is classified as Serious eye damage (category 1). Eye irritation test shows persistence of red staining at the end of the 21 days recovery period which is according to the CLP guideline a reason for Serious eye damage Cat 1 (discoloration of the cornea by a dye substance).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on criteria defined in Regulation (EC) No 1272/2008, RED RWa 4565 is classified as Serious eye damage (category 1). Eye irritation test shows persistence of red staining at the end of the 21 days recovery period which is according to the CLP guideline a reason for Serious eye damage Cat 1 (discoloration of the cornea by a dye substance).