Reaction mass of trisodium [m[7-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-8-hydroxynaphthalene-1,6-disulphonato(7-)]]dicuprate(3-) and trisodium [m[4-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-3-hydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: all animals were housed in single cages.
- Diet: a standard laboratory diet was provided ad libitum. The batch of the diet used for this study was analysed for chemical and microbiological contaminants.
- Water: water was provided ad libitum.
- Acclimation period: all animals were acclimatized to the environment for a period of five days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: the room temperature was maintained within a median of 21 ± 2 °C recorded by a maximum-minimum thermometer.
- Humidity: 30 - 70 %, measured by a hygrometer.
- Air changes: the rate of air exchange was maintained at approximately 15 changes/hour.
- Photoperiod: the light/dark cycle was 12 hours.

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

other: the substance was diluted with WEM (50 %)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

1.0 ml of solution

Duration of treatment / exposure:

4 hours

Number of animals:

Three male rabbits

Details on study design:

TEST SITE
- Area of exposure: 24 hours prior to the test, the skin on the back of the animals was clipped and the skin was investigated for pre-existing injuries. Test item was applied by surgical gauze, measuring 6.5 cm2.
- Type of wrap if used: the animals were immobilized with patches secured in place by a plastic patch and the entire trunk was wrapped with a plastic sleeve.

SCORING SYSTEM
The irritation was measured by patch test technique on the intact skin. After 4 hours, the patches were removed and the resulting reactions were evaluated on the basis of the code of Federal Regulation, Title 16, Section 1500.41.

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:

The irritation score resulted 0.00, thus the substance was considered to be non-irritant.

Individual reactions

Animal	Reaction	30 min	24 hrs	48 hrs	72 hrs	Mean 24/48/72 hrs
Animal 1	Erythema	0	0	0	0	0
Animal 2	Erythema	0	0	0	0	0
Animal 3	Erythema	0	0	0	0	0
Animal 1	Oedema	0	0	0	0	0
Animal 2	Oedema	0	0	0	0	0
Animal 3	Oedema	0	0	0	0	0

Interpretation of results:

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

Conclusions:

Non-irritant

Executive summary:

Three male rabbits were used to assay the skin irritation potential of the test item. 1.0 ml of the solution (test item diluted with WEM (50 %)) were placed on the surgical gauze and place in contact with skin over a period of 4 hours. The irritation was measured by patch test technique on the intact skin. After 4 hours, the patches were removed and the resulting reactions were evaluated on the basis of the code of Federal Regulation, Title 16, Section 1500.41.

The irritation score resulted 0.00, thus the substance was considered to be non-irritant.

Conclusion

Although the criteria for the evaluation of the skin irritation potential followed in the test are different from those reported into the OECD guideline, the scoring system is the same. Because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC) No 1272/2008. The mean values from gradings at 24, 48 and 72 hours were lower than 2.3 for both the erythema and oedema. Thus the substance does not meet the criteria to be classified as skin 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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Housing: all animals were housed in single cages.
- Diet: a standard laboratory diet was provided ad libitum. The batch of the diet used for this study was analysed for chemical and microbiological contaminants.
- Water: water was provided ad libitum.
- Acclimation period: all animals were acclimatized to the environment for a period of five days prior to the start of the study.
- Health check: prior to the test, the eyes of each rabbit were examined for pre-existing casual injury.

ENVIRONMENTAL CONDITIONS
- Temperature: the room temperature was maintained within a median of 21 ± 2 °C recorded by a maximum-minimum thermometer.
- Humidity: 30 - 70 %, measured by a hygrometer.
- Air changes: the rate of air exchange was maintained at approximately 15 changes/hour.
- Photoperiod: the light/dark cycle was 12 hours.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 mg of the test material was placed into the conjunctival sac of the right eyes of the animals.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three male rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: eyes unwashed.

SCORING SYSTEM
The eyes were inspected after 30 min, 24, 48 and 72 hours.
Reading scores for ocular lesions were made according to the recommendations of the USA Food and Drug officials, published in 1959 (Appraisal of the Safet of Chemicals in Foods, Drugs and Cosmetics).

Irritation parameter:

cornea opacity score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

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

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The irritation score resulted 0.67, thus the substance was considered to be non-irritant.

Individual reaction

Animal	Reaction	30 min	24 hrs	48 hrs	72 hrs	mean 24/48/72 hrs
Animal 1	Corneal opacity	0	0	0	0	0.00
Animal 2	Corneal opacity	0	0	0	0	0.00
Animal 3	Corneal opacity	0	0	0	0	0.00
Animal 1	Iris	0	0	0	0	0.00
Animal 2	Iris	0	0	0	0	0.00
Animal 3	Iris	0	0	0	0	0.00
Animal 1	Conjunctival redness	0	0	0	0	0.00
Animal 2	Conjunctival redness	0	1	0	0	0.33
Animal 3	Conjunctival redness	0	2	1	0	1.00
Animal 1	Conjunctival chemosis	0	0	0	0	0.00
Animal 2	Conjunctival chemosis	0	0	0	0	0.00
Animal 3	Conjunctival chemosis	0	0	0	0	0.00

Animal	Findings		30 min	24 hrs	48 hrs	72 hrs
Animal 1	Cornea	(A) density	0	0	0	0
		(B) area	0	0	0	0
		AxBx5	0	0	0	0
	Iris and aspect	(C)	0	0	0	0
		Cx5	0	0	0	0
	Conjuntiva	(D) secretion	0	0	0	0
		(E) swelling	0	0	0	0
		(F) redness	0	0	0	0
		(D+E+F) x 2	0	0	0	0
Animal 2	Cornea	(A) density	0	0	0	0
		(B) area	0	0	0	0
		AxBx5	0	0	0	0
	Iris and aspect	(C)	0	0	0	0
		Cx5	0	0	0	0
	Conjuntiva	(D) secretion	0	0	0	0
		(E) swelling	0	0	0	0
		(F) redness	0	1	0	0
		(D+E+F) x 2	0	2	0	0
Animal 3	Cornea	(A) density	0	0	0	0
		(B) area	0	0	0	0
		AxBx5	0	0	0	0
	Iris and aspect	(C)	0	0	0	0
		Cx5	0	0	0	0
	Conjuntiva	(D) secretion	0	0	0	0
		(E) swelling	0	0	0	0
		(F) redness	0	2	1	0
		(D+E+F) x 2	0	4	2	0

Interpretation of results:

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

Conclusions:

Non-irritant

Executive summary:

Three male rabbits were used to assay the eye irritation potential of the test item. 100 mg of the substance were placed into the conjunctival sac of the right eyes of the animals. Eye were not washed; they were were inspected after 30 min, 24, 48 and 72 hours. Reading scores for ocular lesions were made according to the recommendations of the USA Food and Drug officials, published in 1959 (Appraisal of the Safet of Chemicals in Foods, Drugs and Cosmetics).

The irritation score resulted 0.67, thus the substance was considered to be non-irritant.

Conclusion

Although the criteria for the evaluation of the eye irritation potential followed in the test are different from those reported into the OECD guideline, the scoring system is the same. Because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC) No 1272/2008. 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 all the 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

Three male rabbits were used to assay the skin irritation potential of Direct Blue 251. 1.0 ml of the solution (test item diluted with WEM (50 %)) were placed on the surgical gauze and place in contact with skin over a period of 4 hours. After 4 hours, the patches were removed and the resulting reactions were evaluated on the basis of the code of Federal Regulation, Title 16, Section 1500.41. The irritation score resulted 0.00, thus the substance was considered to be non-irritant.

Although the criteria for the evaluation of the skin irritation potential followed in the test are different from those reported into the OECD guideline, the scoring system is the same. Because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC) No 1272/2008.

EYE IRRITATION

Three male rabbits were used to assay the eye irritation potential of the Direct Blue 251. 100 mg of the substance were placed into the conjunctival sac of the right eyes of the animals. Eye were not washed; reading scores for ocular lesions were made according to the recommendations of the USA Food and Drug officials, published in 1959 (Appraisal of the Safet of Chemicals in Foods, Drugs and Cosmetics). The irritation score resulted 0.67, thus the substance was considered to be non-irritant.

Although the criteria for the evaluation of the eye irritation potential followed in the test are different from those reported into the OECD guideline, the scoring system is the same. Because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC) No 1272/2008.

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.

The mean values from gradings at 24, 48 and 72 hours were lower than 2.3 for both the erythema and oedema, thus the substance does not meet the criteria to be classified as skin irritating.

 

According to the CLP Regulation (EC) No1272/2008, 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. Eye irritation means the production of changes in the eye, which are 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 all the tested animals; all the reactions recorded were fully reversible within 72 hours. 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 for the skin/eye irritation potential, according to the CLP Regulation (EC) No 1272/2008.