Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Description of key information

Acid Blue 225 is to be considered as not irritating to the skin as well as to the eye 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:

04 June 1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according 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)

Deviations:

not specified

GLP compliance:

no

Specific details on test material used for the study:

Name: 21036 / A
Purity: 79.9 %

Species:

rabbit

Strain:

other: Russian Breed

Details on test animals or test system and environmental conditions:

Six rabbits (3 males/3 females) of the Russian breed, weighing 1.5 to 2 kgs, were shaved on the whole back and flanks two days before treatment with an electric clipper. In order to prevent injuries by bites the rabbits were kept separately in V2A wire cages, where they had access to food (NAFAG, Gossau SG, rabbit food) and water ad libitum. The shaven skin on the left side was slightly scarified immediately before treatment. A gauze patch 2.5 x 2.5 cm was soaked with the test substance and immediately applied to the prepared skin. The patch was covered with a plastic film 5 x 5 cm, which was fixed to the body with adhesive tape.

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

other: 50 % polyethylene glycol trituration (PEG 400)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

FAT 21036/A was applied to each side in quantities of 0.5 g.

Duration of treatment / exposure:

The gauze patches were removed 24 hours after the application.

Observation period:

The reaction of the skin was appraised upon removal and 72 hours after it.

Number of animals:

3 males and 3 females

Details on study design:

FAT 21036/A was applied to each side in quantities of 0.5 g. Before application a 50 % polyethylene glycol trituration (PEG 400) was made. The gauze patches were removed 24 hours after the application. The reaction of the skin was appraised upon removal and 72 hours after it.

Evaluation of Skin Reactions
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

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

Irritation parameter:

erythema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Remarks:

scarified skin

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Remarks:

scarified skin

Basis:

mean

Remarks:

6 animals

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 21036/A was found to be not irritating to the rabbit skin.

Executive summary:

A study was performed to determine the skin irritation potential of FAT 21036/A on Russian breed rabbits according to "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO). Six rabbits (3 males/3 females) weighing 1.5 to 2 kgs, were shaved on the whole back and flanks two days before treatment with an electric clipper. In order to prevent injuries by bites the rabbits were kept separately in V2A wire cages, where they had access to food (NAFAG, Gossau SG, rabbit food) and water ad libitum. The shaven skin on the left side was slightly scarified immediately before treatment. A gauze patch 2.5 x 2.5 cm was soaked with the test substance and immediately applied to the prepared skin. The patch was covered with a plastic film 5 x 5 cm, which was fixed to the body with adhesive tape. FAT 21036/A was applied to each side in quantities of 0.5 g. Before application a 50 % polyethylene glycol trituration (PEG 400) was made. The gauze patches were removed 24 hours after the application. The reaction of the skin was appraised upon removal and 72 hours after it. No erythema or edema was observed in the study. All scores were zero. Hence, the primary irritation index as the measure of the acute irritation to the skin of rabbits was found to be 0. Therefore, FAT 21036/A is to be considered as not irritating to the skin of rabbits.

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:

1974

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according 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).

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

Name: 21036 / A
Purity: 79.9%

Species:

rabbit

Strain:

other: Russian Breed

Details on test animals or tissues and environmental conditions:

The substance was tested on 6 rabbits (3 males/3 females) of the Russian breed, which were kept separately in V2A wire cages and fed on NAFAG, Gossau SG, rabbit food and water ad libitum. Only rabbits showing normal ophthalmic findings were included in the test.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g

Duration of treatment / exposure:

Amounts of 0.1 g of the test substance were introduced into the conjunctival sac of the left eye with a spatula. After application, the eyelids were held open for a few seconds. The right eye served as a control. The treated eyes of three rabbits were each rinsed with 10 mL lukewarm water about 30 seconds after treatment.

Observation period (in vivo):

The reactions were appraised with a slit-lamp after 24 hours and after 2, 3, 4 and 7 days on the basis of the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the AFDO.

Number of animals or in vitro replicates:

6 rabbits (3 males/3 females)

Details on study design:

SCORING SYSTEM:

Cornea
A. Opacity-degree of density (area most dense taken for reading)
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


Iris

Normal ...................................................................................................................0
Folds above normal, congestion, swelling, circumcorneal 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

Conjunctivae
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


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

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 adjacent to lids.........................................................................2
Discharge with moistening of the lids and hairs, and considerable area around the eye..............................................3


TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48h

Table: Scores when eyes were not rinsed

Days after application	Rabbit 4			Rabbit 5			Rabbit 6
	Cornea	iris	Conjunctiva	Cornea	iris	Conjunctiva	Cornea	iris	Conjunctiva
1	20	0	12	15	0	12	15	0	12
2	20	0	10	10	0	10	15	0	10
3	15	0	10	10	0	10	10	0	10
4	5	0	8	10	0	8	10	0	6
7	5	0	6	5	0	8	10	0	6

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 21036/A is to be considered as a non-irritant to the eye of rabbits.

Executive summary:

A key study was performed to determine the eye irritation potential of FAT 21036/A in rabbits. The procedure adopted was the technique 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/3 females) of the Russian breed. 0.1 g of the test substance were introduced into the conjunctival sac of the left eye with a spatula. After application, the eyelids were held open for a few seconds. The right eye served as a control. The treated eyes of three rabbits were each rinsed with 10 mL lukewarm water about 30 seconds after treatment. The reactions were appraised with a slit-lamp after 24 hours and after 2, 3, 4 and 7 days. Cornea and Iris scores were zero at all time intervals. slight conjuctival irritation was observed in two rabbits which were reversible within 48 h. The irritation index was found to be 5.8 for the cornea, 0 the iris and 5 for the conjunctivae. According to the EC classification of the results obtained, FAT 21036/A can be classified as non irritant to albino rabbit's eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

The skin irritation potential of FAT 21036/A was assessed in a study conducted according to "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO). Six Russian breed rabbits were exposed to 0.5 g FAT 21036/A each on shaved as well as scarified skin. No erythema or edema was reported throughout the study. The primary irritation index as the measure of the acute irritation to the skin of rabbits was found to be 0. Therefore FAT 21036/A is to be considered as not irritating to the skin of rabbits.

Eye:

A key study was performed to determine the eye irritation potential of FAT 21036/A in rabbits. The procedure adopted was the technique 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/3 females) of the Russian breed. 0.1 g of the test substance were introduced into the conjunctival sac of the left eye with a spatula. After application, the eyelids were held open for a few seconds. The right eye served as a control. The treated eyes of three rabbits were each rinsed with 10 mL lukewarm water about 30 seconds after treatment. The reactions were appraised with a slit-lamp after 24 hours and after 2, 3, 4 and 7 days. Cornea and Iris scores were zero at all time intervals. slight conjuctival irritation was observed in two rabbits which were reversible within 48 h. The irritation index was found to be 5.8 for the cornea, 0 the iris and 5 for the conjunctivae. According to the EC classification of the results obtained, FAT 21036/A can be classified as non irritant to albino rabbit's eye.

Justification for classification or non-classification

Based on the above stated assessment, Acid Blue 225 is to be considered as a non-irritant to the skin as well as to eye of rabbits according to CLP (Regulation (EC) No. 1272/2008 Of the European Parliament and of the Council.