Trisodium [μ-[3-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-4,5-dihydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)

Endpoint summary

Description of key information

Direct Blue 094 is not an irritant to skin and eyes of rabbits.

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:

From 20 Nov, 1978 to 28 Nov, 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: the patch test technique described in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) , the US Association of Food and Drug Officials (AFDO).

Deviations:

yes

Remarks:

Abrasion represent an elevated testing condition compared to todays standard testing conditions accordint to OECD guidelines.

GLP compliance:

no

Remarks:

Study predates GLP

Specific details on test material used for the study:

Batch No: EN 80524/76
Physical appearance: Powder

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Metal cages
- Diet: ad libitum standard rabbit food - NAFAG, Gossau SG
- Water: ad libitum
- Acclimation period: 4 days


ENVIRONMENTAL CONDITIONS
- Temperature: 22±1 °C
- Humidity: 55±5 %
- Light cycle: 10 h light

Type of coverage:

occlusive

Preparation of test site:

other: shaved and abraded

Vehicle:

other:

Remarks:

50% propylene glycol + saline (70:30 parts)

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

24 hours

Observation period:

7 days

Number of animals:

Six rabbits (3 males and 3 females)

Details on study design:

The test was performed on 3 male and 3 female adult rabbits of the Himalayan breed (WIGA Versuchstierzuchtanstalt, 8741 Sulzfeld, Germany) weighing 1.5 to 2 kgs. They were housed individually in metal cages, were kept at a constant room temperature of 22 ± 1 °C, at a relative humidity of 55 ± 5 % and on a 10 hours light cycle day.
The animals received ad libitum standard rabbit food - NAFAG, Gossau SG - and water. Prior to treatment they were adapted to our laboratories for a minimum of 4 days.

Before treatment the entire back and the flank of the rabbits were shaved with an electric clipper and immediately before treatment the shaven skin on one side was slightly scarified with the help of a "SchröpfSchnäpper", Aesculap, Switzerland. Gauze patches of 2.5 x 2.5 cm laden with 0.5 g of the test material were applied to the prepared abraded and intact skin.
The patches were covered with an impermeable material and were fastened to the body of the rabbit with adhesive tape. The dressings were removed after a 24 hour application. The skin reaction was appraised upon removal and during an observation period of 7 days on the basis of the following evaluation scheme.

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

Total possible erythema score .........................................................................................................4

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

Total possible edema score ............................................................................................................4

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 11127/B was considered to be a non-irritant to rabbit skin.

Executive summary:

FAT 11127/B was tested for skin irritation potential according to the patch technique described in "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO)." 0.5 g of test substance was suspended at 50 % concentration in propylene glycol + saline (70 : 30 parts) and applied on the intact as well as abraded skin of 6 Himalayan rabbits (3 males and 3 females) for period of 24 hours. The skin reaction was appraised upon removal and during an observation period of 7 days. Due to intensive staining by the test compound erythema was not assessable. For the purpose of calculating the primary irritation index erythema was assumed to be equal to edema based on the correlation existing (Marzulli and Maibach, Fol. Cosm. Tox. 13, 355, 1975). Following this methodology, it was concluded that no erythema or edema were seen at any of the observations. Hence, FAT 11127/B was considered to be a non-irritant to rabbit skin.

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:

From 20 Nov, 1978 to 28 Nov, 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO).

GLP compliance:

no

Specific details on test material used for the study:

- Batch No.: EN 80524/76
- Physical appearance: Powder

Species:

rabbit

Strain:

other: Russian breed

Details on test animals or tissues and environmental conditions:

The test was performed on 3 male and 3 female adult rabbits of the Himalayan breed (WIGA Versuchstierzuchtanstalt, 8741 Sulzfeld, Germany) weighing 1.5 to 2 kgs. The animals were housed individually in metal cages, were kept at a constant room temperature of 22 ± 1 °C, at a relative humidity of 55±5 % and on a 10 hour light cycle day. The animals had ad libitum access to standard rabbit food - NAFAG, Gossau SG - and water. Prior to treatment they were adapted to our laboratories for a minimum of 4 days. Only rabbits with normal ophthalmic findings were used for these tests.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.1 g

Duration of treatment / exposure:

30 seconds

Observation period (in vivo):

24 hours and after 2, 3, 4 and 7 days

Details on study design:

The test material in an amount of 0.1 g was inserted into the conjunctival sac of the left eye of the rabbits and the lids were gently closed for a few seconds. The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 ml of physiological saline. The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7 and was scored for each individual rabbit.

CORNEA:
A. Opacity and degree of density (most dense area scored)
No opacity 0
Scattered or diffuse area, details of iris clearly visible 1
Easily discernible translucent areas, details of iris slightly obscured 2
Opalescent areas, no details of iris visible, size of pupil barely discernible 3
Opaque, iris invisible 4

B. Area of cornea involved
One quarter (or less) but not zero 1
Greater than one quarter, but less than half 2
Greater than half, but less than three quarters 3
Greater than three quarters, up to whole area 4

A x B x 5 Maximum possible score = 80

IRIS:
A. Values
Normal 0
Folds above normal, congestion, swelling, circumcornea injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive 1
No reaction to light, hemorrhage, gross destruction (any or all of these) 2

A x 5 Maximum possible score = 10

CONJUNCTIVAE :
A. Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal 0
Vessels definitely injected above normal 1
More diffuse, deeper crimson red, individual vessels not easily discernible 2
Diffuse beefy red 3

B. Chemosis
No swelling 0
Any swelling above normal (includes nictitating membrane) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids about half closed to completely closed 4

C. Discharge
No discharge 0
Any amount different from normal (does not include small amounts observed in inner canthus of normal animals) 1
Discharge with moistening of the lids and hairs just adj acent to lids 2
Discharge with moistening of the lids and hairs, and considerable area around the eye 3

(A + B + C) x 2 Maximum possible score = 20

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 11127/B was found to cause no irritation when applied to the rabbit eye mucosa.

Executive summary:

FAT 11127/B was tested for eye irritation potential according to the procedure adopted described in "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO)".

0.1 g of test substance was introduced into conjuctival sac of the left eye of Russian rabbits (3 males and 3 females). The reactions on cornea, iris and conjuctivae were appraised with a slit-lamp after 24 hours and after 2,3,4 and 7 days. No irritation was seen with cornea, iris and conjunctivae at any of the observations. Therefore FAT 11127/B is to be considered to be a non-irritant to rabbit eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Direct Blue 094 has been tested for irritation to skin and eyes in rabbits in two studies each.

In the study designated to be the key study, FAT 11127/B was tested for skin irritation potential according to the patch technique described in "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO)." 0.5 g of test substance was suspended at 50 % concentration in propylene glycol + saline (70 : 30 parts) and applied on the intact as well as abraded skin of 6 Himalayan rabbits (3 males and 3 females) for period of 24 hours. The skin reaction was appraised upon removal and during an observation period of 7 days. Due to intensive staining by the test compound erythema was not assessable. For calculating the primary irritation index erythema was assumed to be equal to edema based on the correlation existing (Marzulli and Maibach, Fol. Cosm. Tox. 13, 355, 1975). Following this methodology, it was concluded that no erythema or edema were seen at any of the observations. Hence, FAT 11127/B is a non-irritant to rabbit skin.

In a similar study, 0.5 g of FAT 11127/A was suspended in 50 % of PEG 400 and applied on the intact and abraded skin of rabbits (3 males and 3 females) by soaking test substance on the gauze patch for a period of 24 hours. The gauze patches were removed 2 4 hours after the application. The reaction of the skin was appraised upon removal and 72 hours after it. No erythema or edema was observed till 72 hours of observation period. Hence, FAT 11127/A was considered a non-irritant to rabbit skin.

Eye irritation:

In the study designated as the key study, FAT 11127/B was tested for eye irritation potential according to the procedure adopted described in "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO)". 0.1 g of test substance was introduced into conjuctival sac of the left eye of Russian rabbits (3 males and 3 females). The reactions on cornea, iris and conjuctivae were appraised with a slit-lamp after 24 hours and after 2,3,4 and 7 days. No irritation was seen with cornea, iris and conjunctivae at any of the observations. Therefore, FAT 11127/B is a non-irritant to rabbit eye.

In a similar study, 0.1 g of FAT 11127/A was introduced into conjuctival sac of the left eye of Russian rabbits (3 males and 3 females). The treated eyes of three rabbits were each rinsed with 10 ml lukewarm water about 30 seconds after treatment. The reactions on cornea, iris and conjuctivae were appraised with a slit-lamp after 24 hours and after 2, 3, 4 and 7 days. The primary-irritation index, which serves as a measure of the acute irritation provoked by the substance, was found to be 0 for the cornea, 0 for the iris and 3.1 for the conjunctivae. According to the AFDO scheme used, the scores for conjunctivae obtained were 6, 8 and 6 at 24 hours for animal# 1, 2 and 3 whose eyes were rinsed 30 seconds after insertion. Further, scores recorded for these animals were 6 (reversed by 72 hours), 0 and 0. For the animal# 4, 5 and 6 whose eyes were not rinsed at any point of time during the study, the scores for conjunctivae were 10 each (24 hours), 6 each (48 hours) and 6 each (72 hours). The reactions were observed to have been reversed by 96 hours. Making reasonable assumption, it can be said that the scores did not meet the criteria of conjunctival redness ≥2 or chemosis ≥2. Hence, no classification is required based on the outcome of the study. Therefore FAT 11127/A is not an irritant to the eyes of rabbits.

 

Based on the above information, Direct Blue 094 is not an irritant to skin and eyes of rabbits and does not need to be classified in this regard.

Justification for classification or non-classification

Direct Blue 094 is concluded to be not an irritant to skin or eyes, hence it does not warrant classification as per the criteria of Regulation (EC) No. 1272/2008.