Disodium 5-(benzoylamino)-4-hydroxy-3-[[2-(2-methylphenoxy)phenyl]azo]naphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Skin irritation: not irritating

Eye Irritation: not 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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test was performed according to internationally accepted testing guidelines

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Remarks:

pre-GLP

Species:

rabbit

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Russian breed
- Weight at study initiation: from 1.4 to 2.3 kg
- Housing: metal cages
- Diet and water: fed with standard diet of Nafag and drinking water ad libitum
- Acclimation period: the animals were acclimatized to the environment in laboratories for at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 1 °C
- Humidity: 55 ± 5 %
- Photoperiod: 14 hours light/day.

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Amount of 0.5 g of test substance were applied

Duration of treatment / exposure:

The gauze patches were removed 24 hours after application

Observation period:

The reaction of the skin was appraised upon removal during an observation period of 8 days

Number of animals:

6 (3 males and 3 females) rabbits

Details on study design:

TEST SITE
- Area of exposure: shaved one day before treatment. The shaved skin area on the left side was slightly scarified immediately before treatment. A gauze patch of 2.5x2.5 cm laden with the test substance was immediately applied to the prepared skin.
- Type of wrap if used: the patch was covered with an impermeable foil of 5x5 cm, which was fixed to the body of the animals with adesive tape.

SCORING SYSTEM: The reaction of the skin was appraised upon removal during an observation period of 8 days.

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

Reversibility:

other: No skin reaction observed

Remarks on result:

other: The complete table of results is attached below

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No skin reaction observed

Remarks on result:

other: The complete table of results is attached below

Irritant / corrosive response data:

The test substance was found to be non irritant when applied to intact or abraded rabbit skin, after an observation period of 8 days. The mean score for each animal is zero.

Interpretation of results:

not classified

Remarks:

Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU

Conclusions:

The test substance was found to be non irritant when applied to intact or abraded rabbit skin.

Executive summary:

METHOD

The skin irritation test was performed to rabbit skin using a single administration, according to the "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).

RESULT/CONCLUSION

The criteria for the evaluation of the skin irritation potential followed in the test are different from those reported into the OECD guideline. Nevertheless, the scoring system used for the erythema and eschar formation and for oedema formation are the same of that reported into the OECD. Since the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC n. 1272/2008).

The test substance was found to be non irritant when applied to intact or abraded rabbit skin.

Therefore, the test substance can be classified as NON IRRITATING, according to the CLP Regulation (EC n. 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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test was performed according to internationally accepted testing guidelines.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Remarks:

pre-GLP

Species:

rabbit

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Russian breed
- Housing: cages
- Diet and water: standard diet of Nafag and drinking water ad libitum.
Only rabbits showing normal ophthalmic findings were included in the test.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 1 °C
- Humidity: 55 ± 5 %
- Photoperiod (hrs dark / hrs light): 14 hours light/day

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

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.

Duration of treatment / exposure:

The eyes of 3 animals were rinsed 1 minute after application

Observation period (in vivo):

8 days

Number of animals or in vitro replicates:

6 (3 males and 3 females) rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): physiologic saline
- Time after start of exposure: 1 minute

TOOL USED TO ASSESS SCORE: hand-slit lamp

SCORING SYSTEM: the reactions were appraised after 1 and 6 hours; 1, 2, 3, 6 and 8 days on the basis of the "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).

CORNEA
- 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
- 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: 2
Swelling with lids about half closed: 3
Swelling with lids about half closed to completely closed: 4
- 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

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not relevant

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not relevant

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not relevant

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritant / corrosive response data:

The substance was found to cause transient minimal to slight irritation in un-rinsed and minimal irritation in rinsed eyes, in both cases after 6 hours. The irritation consisted of conjunctival reaction. Reflective hyperemia of the iris was observed 6 hours after installation in 1/3 unrinsed eyes only.After 24 hours, there was complete recovery in 2/3 unrinsed and 3/3 rinsed eyes.

time		1 hour						6 hours
Eye, left/right		not rinsed			rinsed after 1 minute			not rinsed			rinsed after 1 minute
Animal n.		142	107	143	152	140	191	142	107	143	152	140	191
cornea	opacity	0	0	0	0	0	0	0	0	0	0	0	0
	area involved	0	0	0	0	0	0	0	0	0	0	0	0
iris		0	0	0	0	0	0	0	1	0	0	0	0
conjunctivae	redness	2	2	1	1	2	2	1	2	2	1	1	2
	chemosis	1	2	1	0	1	1	1	2	1	0	0	1
	discharge	2	3	1	1	1	2	1	2	1	0	0	1

time		24 h						48 h
Eye, left/right		not rinsed			rinsed after 1 minute			not rinsed			rinsed after 1 minute
Animal n.		142	107	143	152	140	191	142	107	143	152	140	191
cornea	opacity	0	0	0	0	0	0	0	0	0	0	0	0
	area involved	0	0	0	0	0	0	0	0	0	0	0	0
iris		0	0	0	0	0	0	0	0	0	0	0	0
conjunctivae	redness	1	0	0	0	0	0	0	0	0	0	0	0
	chemosis	0	0	0	0	0	0	0	0	0	0	0	0
	discharge	0	0	0	0	0	0	0	0	0	0	0	0

time		72 h						6 days						8 days
Eye, left/right		not rinsed			rinsed after 1 minute			not rinsed			rinsed after 1 minute			not rinsed			rinsed after 1 minute
Animal n.		142	107	143	152	140	191	142	107	143	152	140	191	142	107	143	152	140	191
cornea	opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	area involved	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
iris		0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
conjunctivae	redness	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	chemosis	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	discharge	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Interpretation of results:

not classified

Remarks:

Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU

Conclusions:

Not irritating

Executive summary:

METHOD

The eye irritation test was performed to rabbit eye using a single administration, according to the "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).

RESULT

The substance was found to cause minimal to slight irritation when applied to the rabbit eye mucosa. The substance was found to cause transient minimal to slight irritation in un-rinsed and minimal irritation in rinsed eyes, in both cases after 6 hours (during the test, the eyes of three animals were rinsed 1 minute after application, with physiologic saline solution). The irritation consisted of conjunctival reaction. Reflective hyperemia of the iris was observed 6 hours after installation in 1/3 unrinsed eyes only. After 24 hours, there was complete recovery in 2/3 unrinsed and 3/3 rinsed eyes.

CONCLUSION

The criteria for the evaluation of the eye irritation potential followed in the test are different from those reported into the OECD guideline. Nevertheless, the scoring system used for the cornea opacity and degree of density, for the iris and conjunctivae redness and chemosis are the same of that reported into the OECD. Since the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC n. 1272/2008).

The mean values at 24, 48 and 72 hours for both rinsed and unrinsed eyes were lower than 1 for corneal opacity, lower than 1 for irtitis, lower than 2 for both conjunctival redness and oedema, in all animals.

Therefore, the test substance can be classified as NON IRRITATING, according to the CLP Regulation (EC n. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation test was performed to rabbit skin using a single administration, according to the "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).

The test substance was found to be non irritant when applied to intact or abraded rabbit skin.

The eye irritation test was performed to rabbit eye using a single administration, according to the "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).

The substance was found to cause minimal to slight irritation when applied to the rabbit eye mucosa. The substance was found to cause transient minimal to slight irritation in un-rinsed and minimal irritation in rinsed eyes, in both cases after 6 hours (during the test, the eyes of three animals were rinsed 1 minute after application, with physiologic saline solution). The irritation consisted of conjunctival reaction. Reflective hyperemia of the iris was observed 6 hours after installation in 1/3 unrinsed eyes only. After 24 hours, there was complete recovery in 2/3 unrinsed and 3/3 rinsed eyes.

The criteria for the evaluation of the eye irritation potential followed in the test are different from those reported into the OECD guideline. Nevertheless, the scoring system used for the cornea opacity and degree of density, for the iris and conjunctivae redness and chemosis is the same of that reported into the OECD. Since the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (1272/2008).

Justification for selection of skin irritation / corrosion endpoint:

The study was performed according to an internationally accepted testing guideline and it met the endpoint requirements fully.

Justification for selection of eye irritation endpoint:

The study was performed according to an internationally accepted testing guideline and it met the endpoint requirements fully.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008) substances that have the potential to induce reversible damage to the skin following the application of a test substance for up to 4 hours are classified in Category 2 (irritating to the skin) if:

- Mean value of ≥ 2.3 ≤ 4.0 for erythema/eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions; or

- Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

- In some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal but less than the criteria above.

In the skin irritation test, the mean values at 24, 48 and 72 hours after patch removal were lower than 2.3 (explicitly, the mean values were zero) in all animals for both erythema/eschar and oedema reactions (in both intact and abraded skin).

According to the CLP Regulation (EC 1272/2008) substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes) if, when applied to the eye of an animal, a substance produces at least in 2 of 3 tested animals, a positive response of:

- corneal opacity ≥ 1 and/or

- iritis ≥ 1, and/or

- conjunctival redness ≥ 2 and/or

- conjunctival oedema (chemosis) ≥ 2,

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

In the eye irritation test, the mean values at 24, 48 and 72 hours for both rinsed and unrinsed eyes were lower than 1 for corneal opacity, lower than 1 for irtitis, lower than 2 for both conjunctival redness and oedema, in all animals.

The available experimental data were considered suitable for a classification and labelling, according to the CLP Regulation (EC 1272/2008). The results show that the substance is not classified for eye and skin irritation.