Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Description of key information

Acid Blue 277 was not irritating to the skin and eyes when tested on 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:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Proposed Guidelines of the United States Environmental Agency (EPA) $ 163.81-5 "Primary dermal irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

Deviations:

yes

Remarks:

(no untreated skin areas to serve as control; exposure period is of 24 h; smaller area of application (i.e., approx. 2.5 cm² gauze pad)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

(no untreated skin areas to serve as control; exposure period is of 24 h; smaller area of application (i.e., approx. 2.5 cm² gauze pad)

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EN 01451.12

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2 to 3 kg
- Housing: The animals were housed individually in metal cages numbered by ear tags
- Diet: Standard rabbit food - NAFAG, No. 814, Gossau SG (ad libitum)
- Water: ad libitum
- Acclimation period: 4 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2 °C
- Humidity: 55±10 %
- Photoperiod (dark / light): 12 h/12 h

Type of coverage:

occlusive

Preparation of test site:

abraded

Vehicle:

other: polyethylene glycol (PEG 400) + saline (70 : 30 parts)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 mL (i.e. 500 mg)
- Concentration (if solution): 50 %

Duration of treatment / exposure:

24 h

Observation period:

7 d

Number of animals:

6 (i.e., 3 males and 3 females)

Details on study design:

TEST SITE:
- 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.

EXPOSURE & OBSERVATIONS:
- Gauze patches of 2.5 x2.5 cm laden with 500 mg of the test substance 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 h application.
- The skin reaction was appraised upon removal and during an observation period of 7 d.

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 4 d

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 3 d

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 4 d

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 3 d

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #5

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 4 d

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 3 d

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 4 d

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 4 d

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #5

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 4 d

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Other effects:

none

Calculation of the primary skin irritation index:

 

MEAN REACTION SCORE
Time after exposure hours	e r y t h e m a		edema
	intact skin	abraded skin	intact skin	abraded skin
24	1.3	1.3	1.3	1.3
72	0.5	0.7	0.5	0.7
Total	1.8	2.0	1.8	2.0	7.6

Primary irritation index = 7.6 : 4 = 1.9

 

Due to intensive staining by the test substance 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.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 20033/G was considered to be slightly irritating when applied to intact rabbit skin, however does not meet the EU CLP threshold for classification.

Executive summary:

An in vivo study was conducted to evaluate the skin irritation potential of the test substance (of ca. 82.2 % purity) in rabbits according to USEPA Guideline 163.81-5 which is similar to OECD Guideline 404 with few deviations. In this study, gauze patches of 2.5 x 2.5 cm laden with 0.5 g of the test material (i.e. 1 mL of a 50 % solution in polyethylene glycol (PEG 400) and saline (70 : 30 parts)) were applied to the prepared abraded and intact skin of rabbits for 24 h under occlusive conditions. After 24 h, the dressing was removed and the application sites were assessed for oedema and erythema. Further observations were conducted at 48, 72 h, 4 and 7 d. The exposure of FAT 20033/G to intact skin lead to slight irritation in terms of erythema and edema, however the scores obtained were below the mean critical threshold of 2.3 for all the exposed animals. The primary irritation score was calculated to be 1.9. Under the study conditions, the test substance was considered to be slightly irritating when applied to intact rabbit skin. However, responses were confirmed as not exceeding the EU CLP thresholds for triggering classification of skin irritant.

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:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EN 01451.12

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2 to 3 kg
- Housing: The animals were housed individually in metal cages
- Diet: Standard rabbit food - NAFAG, No. 814, Gossau SG (ad libitum)
- Water: ad libitum
- Acclimation period: 4 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2 °C
- Humidity: 55±10 %
- Photoperiod (dark / light): 12 h/12 h

Vehicle:

unchanged (no vehicle)

Controls:

other: the second eye on the treated animals served as control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 100 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

Unwashed eye: Duration of treatment same as observation period.
Washed eye: 30 seconds

Observation period (in vivo):

7 d

Number of animals or in vitro replicates:

6 (3 males and 3 females)

Details on study design:

PROCEDURE:
- Only rabbits with normal ophthalmic findings were used for the test.
- The test substance in an amount of 100 mg 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 approx. 30 seconds after treatment the treated eye was flushed with 10 mL of physiological saline.
- The eye irritation was evaluated with a slit-lamp on 1, 2, 3, 4 and 7 d and was scored for each individual rabbit.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with 10 ml of physiological saline
- Time after start of exposure: 30 sec

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

B: Area of cornea involved
1 - One quarter (or less) but not zero
2 - Greater than one quarter, but less than half
3 - Greater than half, but less than three quarters
4 - Greater than three quarters, up to whole area
A x B x 5 Maximum possible score = 80

IRIS
A: Values
0 - Normal
1 - 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)
2 - No reaction to light, hemorrhage, gross destruction (any or all of these)
A x 5 Maximum possible score = 10

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

B: Chemosis
0 - No swelling
1 - Any swelling above normal (includes nictitating membrane)
2 - Obvious swelling with partial eversion of lids
3 - Swelling with lids about half closed
4 - Swelling with lids about half closed to completely closed
C Discharge
0 - No discharge 0
1 - Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2 - Discharge with moistening of the lids and hairs just adjacent to lids
3 - Discharge with moistening of the lids and hairs, and considerable area around the eye
(A + B + C) x 2 Maximum possible score = 20


TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

not fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

not fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

other: Test substance was washed after 30 seconds of application

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: Test substance was washed after 30 seconds of application (however, observation period was up to 7 d only)

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

other: Test substance was washed after 30 seconds of application

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 7 d

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

not fully reversible within: 7 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

not fully reversible within: 7 d

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: Test substance was washed after 30 seconds of application.

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

not fully reversible within: 7d

Remarks on result:

other: Test substance was washed after 30 seconds of application (however, observation period was up to 7 d only)

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: Test substance was washed after 30 seconds of application.

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

Irritant / corrosive response data:

The test substance was found to cause a minimal irritation (i.e., primary irritation index 5.7) when applied to the rabbit eye mucosa. When the application site was rinsed, the irritation was found to be minimal (i.e., primary irritation index 0.7).

Other effects:

none

Calculation of the primary eye irritation index:

	MEAN REACTION SCORE
Time after exposure (d)	Unrinsed eyes (A)			Rinsed eyes (B)
	cornea	iris	conjunctiva	cornea	iris	conjunctiva
1	0	0	8	0	0	1.3
2	0	0	6.7	0	0	1.3
3	0	0	5.3	0	0	0.7
4	0	0	5.3	0	0	0
7	0	0	3.3	0	0	0
subtotal	0	0	28.6	0	0	3.3
total	28.6			3.3

Primary irritation index in unrinsed eyes:

A = 28.6 : 5 = 5.7

Primary irritation index in rinsed eyes:

B = 3.3 : 5 = 0.7

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance produced only slight conjunctival reaction.

Executive summary:

An in vivo study was conducted to evaluate the acute eye irritation potential of the test substance (of ca. 82.2% purity) according to a procedure similar to OECD Guideline 405 with few deviations. 100 mg of the test substance was placed into the conjunctival sac of the left eye of rabbits (4 male and 2 females). The right eye remained untreated and was used as untreated control. In three rabbits washing was done with 10 mL of physiological saline after 30 seconds of test substance instillation. Assessment of ocular damage/irritation was made with a slit-lamp approximately on 1, 2, 3, 4 and 7 d following treatment. No corneal or iridial effects were noted during the study. Minimal irritation was observed with primary irritation index of 5.7 and 0.7 for rinsed and unrinsed eyes respectively. Under the study conditions, the test substance produced only slight conjunctival reaction.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

An in vivo study was conducted to evaluate the skin irritation potential of FAT 20033/G (of ca. 82.2 % purity) in rabbits according to USEPA Guideline 163.81-5 which is similar to OECD Guideline 404 with few deviations. The exposure of FAT 20033/G to intact skin lead to slight irritation in terms of erythema and edema, however, scores obtained were below the mean critical threshold of 2.3 for all the exposed animals. The primary irritation score was calculated to be 1.9. FAT 20033/G was slightly irritating when applied to intact rabbit skin, but does not meet the EU CLP threshold for classification. This outcome was further supported in two irritation studies conducted with FAT 20033/C (1975) and FAT 20033/D (1977).

 

Eye:

An in vivo study was conducted to evaluate the acute eye irritation potential of FAT 20033/G (of ca. 82.2 % purity) according to a procedure similar to OECD Guideline 405 with few deviations. 100 mg of the test substance was placed into the conjunctival sac of the left eye of rabbits (4 male and 2 females). The right eye remained untreated and was used as untreated control. In three rabbits washing was done with 10 mL of physiological saline after 30 seconds of test substance instillation. Assessment of ocular damage/irritation was made with a slit-lamp approximately on 1, 2, 3, 4 and 7 d following treatment. No corneal or iridial effects were noted during the study. Minimal irritation was observed with primary irritation index of 5.7 and 0.7 for rinsed and unrinsed eyes respectively. Under the study conditions, the test substance produced only slight conjunctival reaction. This outcome was further supported in two irritation studies conducted with FAT 20033/C (1975) and FAT 20033/D (1977).

Justification for classification or non-classification

Based on the available information 1-amino-4-({3-[(2-hydroxyethyl)sulfamoyl]-4,5-dimethylphenyl}amino)-9,10 -dioxo-9,10-dihydroanthracene-2-sulfonic acid, sodium salt the substance does not have to be classified as irritating to skin or eyes according to CLP Regulation (EC) No 1272/2008 of the European Parliament and of the Council.