A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

EC Number:

423-940-7

EC Name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

Cas Number:

85585-91-7

IUPAC Name:

4-[2-(7-{2-[4-(ethenesulfonyl)-2-hydroxy-5-methoxyphenyl]diazen-1-yl}-8-hydroxy-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid 4-[2-(8-hydroxy-7-{2-[2-hydroxy-4-(2-hydroxyethanesulfonyl)-5-methoxyphenyl]diazen-1-yl}-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid dicopper tetrasodium hydride

Test material form:

solid

Specific details on test material used for the study:

Identification: Pacified Reactive Black 31
Description: Dark blue crystals
Batch: 9T-55
Purity: 89%
Storage: At room temperature in the dark
Stability in vehicle: Stable in water for at least 96 hours

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Age at start of treatment
Approximately 5 weeks

Body weight at start of treatment
1546 - 1659 h=g

Identification
Ear tag

Conditions
Air conditined room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, ut were considered not to have affected study integruty. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Accomodation
Individually in labelled cages with perforated floors and equipped with an automatic drinking system. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

diet
Standard laboratory rabbit diet, approximately 100 g per day. Certificates of analysis were examined and retained in the NOTOX archives. In addition, hay was provided once a week.

Water
Free access to tap water diluted with decalcifued water.

Certificates of analysis were examined and retained in the NOTOX archives.

Test system

Vehicle:

other: Moistened with water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

78 mg into one eye (approx 0.1 ml)

Duration of treatment / exposure:

Seconds

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3 animals

Details on study design:

The animals were examined, and the eyes in particular, prior to test substance administration and no abnormalities were detected.

On test Day 1, 78 mg (0.1 ml) of the test substance was instilled in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently helfd together for about one second to prevent the loss of the test substance. The other eye remained untreated and served as the reference control.

Immediately after the 24 hour observation, a solution of 2% fluroscein in water (adjusted to pH 7) was instilled in both eyes of each animal to quantitatively determine corneal epithelial damage. any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area,

Immediately after fluroscein examination on Day 2, both eyes of all three animals were rinsed with approximately 50 ml tepid tap water per eye, using a velocity of flow which did not effect the eyes., to remove residual test substance.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

not fully reversible within: 7 days

Results 4

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Results 5

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Results 6

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Instillation of approximately 78 mg of Pacified Reactive Black 31 into one eye of each of three animals resulted in effects on the conjunctivae. Irritation of the conjunctivae consisted of redness, chemosis and discharge, which had resolved in all animals within 7 days after instillation. However, black staining of the conjunctivae by the test substance made scoring for the redness impossible on Day 1. At the following observations, the staining had partly disappeared. Black staining of the nictitating membrane and the eyelid in the lower eye sac persisted until Day 21 oafter instillation.

Iridic irritation grade 1 was noted in one animal and had resolved within 24 hours after instillation.

Treatment of the eyes with 2% fluroscein , 24 hours after test substance instillation revealed no corneal epithelial damage to any of the animals.

There was no evidence of ocular corrosion.

Other effects:

Black staining on the head and paws and of the conjunctivae, caused by the test substance, was noted during the observation period.

Any other information on results incl. tables

No symptoms of sytemic toxicity were observed in the animals during the test period and no mortality occurred.

Applicant's summary and conclusion

Interpretation of results:

other: Mildly irritating

Conclusions:

Pacified Reactive Black 31 was considered mildly irritating (M2) to the rabbit eye (Kay and Calandra interpretation of the calculated maximum Draize score 9.3).

Executive summary:

The study was carried out in accordance with the OECD guideline No. 405, "Acute Eye Irritation/ Corrosion" and the EEC Directive 92/69/EEC, B.5, "Acute Toxicity - Eye Irritation".

Single samples of approximately 78 mg of Pacified Reactive black 31 (a volume of approximatelt 0.1 ml) were instilled into one eye of each of three rabbits. O=bservations were made 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation.

Instillation of Pacified Reactive Black 31 resulted in effects on the conjunctivae, which had resolved in all animals within 7 days after instillation. However, black staining of the conjunctivae by the test substance made scoring for redness impossible on Day 1. At the following obsevrations, the staining had partly disappeared. Blacl staining of parts of the conjunctival tissues persisted until 21 days after instillation.

Iridic irritation was noted in one animal and had resolved within 24 hours after instillation.

Black staining of the fur on the head and paws and of the conjunctivae, caused by the test substance, was noted during the observation period.

Pacified Reactive Black 31 was considered mildly irritating  (M2) to the rabbit eye (Kay and Calandra interpretation of the calculated maximum Draize score 9.3).