Reaction mass of Cobaltate(2-), [2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)][2-[[2-hydroxy-5-[(phenylamino)sulfonyl]phenyl]azo]-3-oxo-N-phenylbutanamidato(2-)]-, disodium and Cobaltate(3-), bis[2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)]-, trisodium and sodium bis[2-[[2-hydroxy-5-[(phenylamino)sulphonyl]phenyl]azo]-3-oxo-N-phenylbutyramidato(2-)]cobaltate(1-)

Endpoint summary

Description of key information

Not skin irritating

Not eye irritating

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:

November from the 01st to the 08th, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

May 12, 1981

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland.
- Age at study initiation: 15 - 16 weeks.
- Weight at study initiation: 2.4 - 3.4 kg
- Housing: individually in stainless cages equipped with an automatic cleaning and drinking system.
- Diet: pelleted standard Kliba 341, Batch 10/85 rabbit maintenance diet ("KIiba" Klingentalmuehle AG, Switzerland), ad Iibitum.
- Water: community tap water from Itigen, ad libitum.
- Acclimation period: four days under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Relative humidity: 55 ± 10 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hrs dark / 12 hrs artificial fluorescent Iight.
- Other: music/light period.

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

2 maels and 1 female

Details on study design:

TEST SITE
- Area of exposure: approximately 24 hours before treatment, the dorsal fur was shaved with an electric clipper, exposing an area of approximately 100 square centimeters (10 cm x 10 cm). It was covered with a 3 0 cm x 3.0 cm patch of surgical gauze.
- Type of wrap if used: t he gauze was covered with aluminium foil and then covered with an occlusive-dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage.

OBSERIVATIONS
- Viability / mortality: daily.
- Bodyweights: Day 1 and at termination of test on a Mattler PK 16 balance.

OBSERVATION TIME POINTS
For each individual animal, the irritation scores for erythema and edema were recorded at 60 minutes, 24, 48 and 72 hours after patch removal; in addition, any lesions, a description of the degree and nature of irritation, corrosion or reversibility and any toxic effect observed were recorded.

SCORING SYSTEM
Four hours after the application, the dressing was removed and the skin was flushed with luke warm tap water. The skin reaction was assessed according to the OECD guideline 404. The skin reaction was assessed at 1-, 24-, 48 and 72 hours intervals after the removal of the dressing, gauze patch and test articIe.
The corrosive properties of the test article and the colour to the treated skin area were described and recorded.

Erythema and Eschar Formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4

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

Irritation parameter:

erythema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2.3

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2.3

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Under the conditions of the experiment, the test item was found to cause a primary irritation score of 0 when applied to intact rabbit skin. In the area of application a clear visible yellowish-brown discoloration of the skin was observed which could be related to effects of the test article.
No destructions or irreversible alterations effect had occurred on the skin at each measuring interval.

Other effects:

BODY WEIGHTS
The body weight gain of all rabbits was similar.

TOXIC SYMPTOMS / MORTALITY
No acute toxic symptoms were observed in the animals during the test period and no mortality occurred.

Animal N. / sex	Reaction	Observation at
		1 hr	24 hrs	48 hrs	72 hrs
298 M	Erythema	0	0	0	0
299 M	Erythema	0	0	0	0
300 F	Erythema	0	0	0	0
298 M	Oedema	0	0	0	0
299 M	Oedema	0	0	0	0
300 F	Oedema	0	0	0	0

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Not irritating

Executive summary:

The purpose of the primary skin irritation study was to assess possible irritation potential when single doses of test item are placed on the skin of rabbits. The experiment was conducted in accordance with the method and procedures outlined into the OECD guideline 404.

Under the conditions of the experiment, the test item was found to cause a primary irritation score of 0 when applied to intact rabbit skin. In the area of application a clear visible yellowish-brown discoloration of the skin was observed which could be related to effects of the test article.

No corrosion effect had occurred on the skin at each measuring interval.

Conclusion

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions, thus the test item does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC) No 1272/2008.

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:

November from the 01st to the 08th, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

May 12, 1981

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland.
- Age at study initiation: 15 - 16 weeks.
- Weight at study initiation: 2.4 - 2.9 kg
- Housing: individually in stainless cages equipped with an automatic cleaning and drinking system.
- Diet: pelleted standard Kliba 341, Batch 10/85 rabbit maintenance diet ("KIiba" Klingentalmuehle AG, Switzerland), ad Iibitum.
- Water: community tap water from Itigen, ad libitum.
- Acclimation period: four days under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Relative humidity: 55 ± 10 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hrs dark / 12 hrs artificial fluorescent Iight.
- Other: music/light period.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Application volume: 0.1 g per animal

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

1 male and 2 females

Details on study design:

OBSERIVATIONS
- Viability / mortality: daily.
- Bodyweights: Day 1 and at termination of test on a Mattler PK 16 balance.

TOOL USED TO ASSESS SCORE: slit-lamp 30 SL.

OBSERVATION TIME POINTS
For each individual animal, the irritation scores for erythema and edema were recorded at 60 minutes, 24, 48 and 72 hours after administration; in addition, any lesions, a description of the degree and nature of irritation, corrosion or reversibility and any toxic effect observed were recorded.

SCORING SYSTEM
Cornea Grade
Opacity: degree of density (readings should be taken from most dense area)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible 1
Easily discernible translucent area; details of iris slightly obscured 2
Nacrous area; no details of iris visible; size of pupil barely discernible 3
Opaque cornea; iris not discernible through the opacity 4

Iris
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is positive) 1
Hemorrhage, gross destruction, or no reaction to light 2

Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)
Normal 0
Some blood vessels hyperaemic (injected) 1
Diffuse,crimson colour; individual vessels not easily discernible 2
Diffuse beefy red 3

Swelling (refers to lids and/or nictating membranes)
Normal 0
Some swelling above normal 1
Obvious swelling, with partial eversion of lids 2
Swelling, with lids about half closed 3
Swelling, with lids more than half closed 4

Irritation parameter:

cornea opacity score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

< 1

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Irritant / corrosive response data:

Under the conditions of the experiment, the test item was found to cause a primary irritation score of 2.4 when applied to rabbit eye mucosa.
In the area of application a slight yellow discoloration of the cornea and conjunctivae was observed which could be related to effects of the test article.
No corrosion effect had occurred on the skin at each measuring interval.

Other effects:

BODY WEIGHTS
The body weight gain of all rabbits was similar.

TOXIC SYMPTOMS / MORTALITY
No acute toxic symptoms were observed in the animals during the test period and no mortality occurred.

Animal N. / sex	Reaction	Observation at				Mean 24/48/72 hrs
		1 hr	24 hrs	48 hrs	72 hrs
295 M	Cornea opacity	1	1	1	0	0.67
296 F	Cornea opacity	1	1	1	1	1.00
297 F	Cornea opacity	0	0	0	0	0.00
295 M	Iris	0	0	0	0	0.00
296 F	Iris	0	0	0	0	0.00
297 F	Iris	0	0	0	0	0.00
295 M	Conjunctival redness	2	1	1	1	1.00
296 F	Conjunctival redness	2	2	1	1	1.33
297 F	Conjunctival redness	2	1	0	0	0.33
295 M	Conjunctival chemosis	3	1	0	0	0.33
296 F	Conjunctival chemosis	2	1	0	0	0.33
297 F	Conjunctival chemosis	1	0	0	0	0.00

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Not irritating

Executive summary:

The purpose of the primary eye irritation study was to assess possible irritation potential when single doses of test item are placed in the conjunctival sac of rabbit eyes. The experiment was conducted in accordance with the method and procedures outlined into the OECD guideline 405.

Under the conditions of the experiment, the test item was found to cause a primary irritation score of 2.4 when applied to rabbit eye mucosa. In the area of application a slight yellow discoloration of the cornea and conjunctivae was observed which could be related to effects of the test article.

No corrosion effect had occurred on the skin at each measuring interval.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in at least 2 out of three tested animals. Thus, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

SKIN IRRITATION

The primary skin irritation potential of the substance was assessed in rabbits. The experiment was conducted in accordance with the method and procedures outlined into the OECD guideline 404. Under the conditions of the experiment, the test item was found to cause a primary irritation score of 0 when applied to intact rabbit skin. In the area of application a clear visible yellowish-brown discoloration of the skin was observed which could be related to effects of the test article. No corrosion effect had occurred on the skin at each measuring interval.

Since the Acid Yellow 235 lot tested in the key study was characterized by an appreciable non-hazardous impurity content, the available data on structural analogous Similar Substance 01 has been taken into account, in order to confirm the study outcomes. The read across approach can be considered as reliable and adequate for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.2.

An in vivo test was carried out for to assess the primary irritation potential of Similar Substance 01, in rabbits. The method followed was patch test technique as described in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO). 6 rabbits (3 male and 3 female) were observed for 7 days; the evaluation was conducted on both intact and scarified skin. The test item was applied to each side in quantities of 0.5 g. Under the tested conditions, the test item was found to be non irritant when applied to the intact or abraded rabbit skin.

EYE IRRITATION

The primary eye irritation potential of the substance was assessed in rabbits. The experiment was conducted in accordance with the method and procedures outlined into the OECD guideline 405. Under the conditions of the experiment, the test item was found to cause a primary irritation score of 2.4 when applied to rabbit eye mucosa. In the area of application a slight yellow discoloration of the cornea and conjunctivae was observed which could be related to effects of the test article. No corrosion effect had occurred on the skin at each measuring interval.

Since the Acid Yellow 235 lot tested in the key study was characterized by an appreciable non-hazardous impurity content, the available data on structural analogous Similar Substance 01 has been taken into account, in order to confirm the study outcomes. The read across approach can be considered as reliable and adequate for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.2.

A study was performed to determine the eye irritation potential of the Similar Substance 01, according to the method and procedures described in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO). The substance was tested on 6 rabbits (3 males and 3 females). Amounts of 0.1 g of the test substance were administrated and the eyes of 3 animals were rinsed 1 minute after application with physiologic saline. Under the tested conditions the substance resulted to be practically non irritant when applied to the rabbit eye mucosa, both in the cases of rinsed eyes and in the cases of non-rinsed eyes.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008) 3.2 Skin corrosion/irritation section, skin irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions, thus the test item does not meet the criteria to be classified as irritating.

According to the CLP Regulation (EC) No 1272/2008, serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application.

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in at least 2 out of three tested animals. Thus, the substance does not meet the criteria to be classified as eye irritating.

In conclusion, the substance does not meet the criteria to be classified either for the skin, neither for the eye irritation, according to the CLP Regulation (EC) No 1272/2008.