Reaction products of diazotised 2-amin-6-nitrophenol-4-sulphonic Acid and 2-Amino-4-nitrophenol-6-sulfonic Acid, coupled with a mixture of 6-Amino-4-hydroxy-2-naphthalenesulfonic Acid and Acetylated 6-Amino-4-hydroxy-2-naphthalenesulfonic Acid, subsequently metallized with Chromium (III) Basic Sulfate and Acetic Acid, and at the end diazotised and coupled with p-Cresol, sodium salts

Eye irritation

Administrative data

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

February 24, 1987

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

405-750-6

EC Name:

-

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

diiron(3+) dihydrogen tris(7-[2-(2-hydroxy-5-methylphenyl)diazen-1-yl]-2-[2-(3-nitro-2-oxido-5-sulfophenyl)diazen-1-yl]-3-sulfonaphthalen-1-olate) 7-acetamido-2-[2-(3-nitro-2-oxido-5-sulfophenyl)diazen-1-yl]-3-sulfonaphthalen-1-olate

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF-Quality

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
-Source: Broekman Institute, Someren, The Netherlands
-Age at start of treatment: approx. 19 weeks
-Body weight at start of treatment: 2703-2906 g
-Identification: ear tag
-Acclimatisation: at least five days under test conditions
-Housing: individual cages with perforated floors equipped with an automatic drinking system (ITL, Bergen / The Netherlands).

ENVIRONMENTAL CONDITIONS
-Temperature: 21 ± 3°C
-Humidity: 30-70 %
-Air changes: 7.5-15 air changes per hr with air-conditioned
-Photoperiod: 12 hrs artificial fluorescent light / 12 hrs dark.
-Diet: standard laboratory rabbit diet (LKK-20, pelleted, Hope Farms, Woerden / The Netherlands), approx. 100 g per day
-Water: tap-water diluted with decalcified water, ad libitum.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
-Amount applied: 78.6 ± 1.0 mg (equivalent to 0.1 ml) / animal
-Preparation: ground to a powder prior to administration.

Duration of treatment / exposure:

single apllication to the conjunctival sac of the left eye of each animal

Observation period (in vivo):

1, 24, 48 and 72 h and 7 d after instillation of the test substance

Number of animals or in vitro replicates:

3 females

Details on study design:

TREATMENT
Prior to test substance administration, on day 1, the animals were physically examined and the eyes found to be intact and normal.
During the substance application the lower lid was gently pulled away from the eyeball. Thereafter the lids were then gently held together for about one second to prevent loss of the test substance.
Immediately after the 24 h observation, a solution of 2% fluorescein in water (pH adjusted to 7.0) was applied to both eyes of each animal to quantitatively examine the potential for corneal injury. The bright green stained area indicating epithelial damage was estimated as a percentage of the total corneal area. In case standard lighting was considered inadequate for observing minor effects, eye examinations were performed using a pocket flash light.

OTHER OBSERVATIONS
-Viability / Mortality, daily.
-Body Weight, once (application day)
-Staining of the treated eye was described and recorded at each observation.

SCORING SYSTEM
CORNEA
-Opacity: degree of density (area most dense taken for reading)
No ulceration or opacity: 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible: 1
Easily discernible translucent area, details of iris slightly obscured: 2
Nacreous areas, no details of iris visible, size of pupil barely discernible: 3
Opaque cornea, iris not discernible through the opacity: 4
-Area of cornea affected
No ulceration or opacity: 0
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
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or any combination thereof, iris still reacting to light (sluggish reaction is positive): 1
No reaction to light, haemorrhage, gross destruction (any or all of these): 2
CONJUNCTIVAE
-Redness: (refers to palpebral and bulbar conjunctivae, excluding cornea and iris).
Blood vessels normal
Some blood vessels definitely hyperaemic (injected): 1
Diffuse, crimson colour, individual vessels not easily discernible: 2
Diffuse beefy red: 3
-Chemosis: lids and/or nictating membrane
No swelling: 0
Any swelling above normal (includes nictating membrana): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids more than half 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, in a considerable area around the eye: 3.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Results 2

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 h

Results 4

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 h

Results 5

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Results 6

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

fully reversible within: 7 d

Results 8

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 h

Results 9

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Results 10

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Results 11

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 7 d

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

IRRITATION
Instillation of the test substance into one of the eyes of each of three albino rabbits affected the conjunctivae. The irritation of the conjunctivae was reversible within 72 h in one animal and within 7 d in the other two animals.
Lacrimation was observed in all animals approximately 1 h after instillation of the test substance.
Treatment of the eyes with fluorescein 2%, 24 h after test substance instillation revealed no corneal epithelial damage in any of the animals.
CORROSION
No corrosion was observed.

Other effects:

TOXIC SYMPTOMS / MORTALITY
No toxic symptoms were observed in the animals during the test period and no mortality occurred.
COLOURATION
Black staining of the conjunctivae by the test substance was observed.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to CLP Regulation (EC 1272/2008)

Conclusions:

The test substance was found to be not irritant when applied to intact rabbit eye.

Executive summary:

The eye irritation test was performed to rabbit eye according to the OECD Guideline 405 (1987). Under the conditions of this study, the substance resulted in a black staining and an adverse effect on the conjunctivae: mean values at 24, 48 and 72 h were 1, 1.7 and 1.3 for redness and 0.3 in all animals for chemosis. These effects were reversible within 72 h in one animal and within 7 d in the other two animals. The mean values at 24, 48 and 72 h for corneal opacity and iris were 0 in all animals. Neither corrosion nor signs of systemic intoxication were observed.