reaction mass of sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium 1-amino-4-[[3-[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

other: data on Acid Blue 225_constituent 1

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

Justification for type of information:

The experiment was conducted on one of the substance components. It should be noted that the lot tested was characterized by the Acid Blue 225_constituent 1 as main component; however, the impurity profile (which also includes the Acid Blue 225_constituent 2) resulted to be closely similar to that characterizing the substance under assessment (details are given in the document attached to IUCLID section 13.2).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1974

Report date:

1974

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

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).

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acid Blue 225_constituent 1

IUPAC Name:

Acid Blue 225_constituent 1

Test animals / tissue source

Species:

rabbit

Strain:

other: Russian Breed

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Housing: rabbits were kept separately in V2A wire cages.
- Diet: food (NAFAG, Gossau SG, rabbit food), ad libitum.
- Water: ad libitum.
- Animal selection: only rabbits showing normal ophthalmic findings were included in the test.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g

Duration of treatment / exposure:

about 30 seconds in 3 rabbits

Number of animals or in vitro replicates:

3 males and 3 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: the treated eyes of three rabbits were each rinsed with 10 ml lukewarm water.
- Time after start of exposure: about 30 seconds after treatment.

TOOL USED TO ASSESS SCORE: slit-lamp

SCORING SYSTEM
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.

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
A x B x 5 Total maximum = 80

Iris
A. Values
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
A x 5

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 adjacent to lids 2
Discharge with moistening of the lids and hairs, and considerable area around the eye 3
(A + B + C) x 2 Total maximum = 20

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

rinsed eye

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

iris score

Remarks:

rinsed eye

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

conjunctivae score

Remarks:

rinsed eye

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 24 hours

Results 4

Irritation parameter:

chemosis score

Remarks:

rinsed eye

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 24 hours

Irritant / corrosive response data:

The irritation index was found to be 5.8 for the cornea, 0 for the iris and 5 for the conjunctivae.

Any other information on results incl. tables

Scores when eyes were rinsed

Days after application	Rabbit 1, female			Rabbit 2, female			Rabbit 3, female
	Cornea	Iris	Conjunctiva	Cornea	Iris	Conjunctiva	Cornea	Iris	Conjunctiva
1	0	0	6	0	0	0	0	0	6
2	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0
7	0	0	0	0	0	0	0	0	0

Scores when eyes were not rinsed

Days after application	Rabbit 4, male			Rabbit 5, male			Rabbit 6, male
	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

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Non irritating

Executive summary:

A study was performed to determine the eye irritation potential of test item 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 3 male and 3 female rabbits. Amounts of 0.1 g of the test substance were introduced into the conjunctival sac of the left eye with a spatula. The treated eyes of three rabbits were each rinsed with 10 ml lukewarm water about 30 seconds after treatment.

The total maximum is 80 for the cornea, 10 for the iris and 20 for the conjunctivae. The irritation index was found to be 5.8 for the cornea, 0 the iris and 5 for the conjunctivae.

Discussion and conclusion

Although the criteria to calculate the irritating potential are different, the scoring system used to record the severity of the eye reactions followed in the current test are the same as those reported into the OECD guideline.

The mean values from gradings at 24, 48 and 72 hours for rinsed eyes can be calculated to be 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.

Regarding the effects on non-rinsed eyes, the mean from gradings at 24, 48, 72 hours cannot be calculated, because raw table are not available. It should be noted that not rinsing the treated eyes could lead to increased irritation response due to the powder form of the substance. Thus, the effects on non-rinsed eyes have not been considered for the classification purpose.