Reaction mass of trisodium 4-amino-3-[[4-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and trisodium 4-amino-3-[[4-[[4-[(4-amino-2-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

October from 18th to October 25th, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The test was conducted by means of Read Across approach. Further information was attached at section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted February 24th, 1987

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Direct Black 168:1 - Similar Substance 01

IUPAC Name:

Direct Black 168:1 - Similar Substance 01

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation:15 weeks males, 16 weeks females.
- Weight at study initiation: 2.6 - 2.8 kg males, 2.1 kg females.
- Housing: individually in stainless steel cages equipped with an automatic cleaning and drinking system.
- Diet: pelleted standard Kliba 341, rabbit maintenance diet, ad libitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation: four days under laboratory conditions after veterinary examination.
- Health check: the eyes of the animals were examined once, one day prior to test article administration. Only those animals with no signs of ocular injury or irritation were used on the test.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark.
- Other: music during the light period, room was air-conditioned.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

- Amount applied: 0.1 g
- Application: the test article was applied undiluted as delivered by the sponsor.

Observation period (in vivo):

72 hours after administration

Number of animals or in vitro replicates:

2 males and 1 female

Details on study design:

APPLICATION OF TEST MATERIAL
The test article was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test article.

REMOVAL OF TEST SUBSTANCE
- Washing: eyes were not rinsed.

OBSERVATIONS
- Viability/Mortality and clinical signs: daily.
- Body Weights: at the start of acclimatization, day 1 of test (application day) and at termination of observation.
- Sacrifice: all rabbits were euthanized by an intravenous injection of T61 (Hoechst/AG) into the ear vein and discarded.

TOOL USED TO ASSESS SCORE: eye examinations were made with a slit-lamp 30 SL/M and a Varta Cliptrix diagnostic-lamp.

SCORING SYSTEM
The eyes of each animal were examined for ocular irritation 1, 24, 48 and 72 hours after administration. The irritation was assessed according to the following numerical scoring system. The corrosive properties of the test article and the staining of the treated eye were described and recorded.
To calculate the irritation effect all scores of each animal at each of the following reading times (24, 48, 72 hours) were used in calculating the respective mean values (Council Directive 83/467/EEC, July 1983 and 67/548/EEC, May 1987, Brussels, Belgium).
The ocular irritation scores were evaluated in conjunction with the nature and reversibility or otherwise of the responses observed.

CORNEAL IRRITATION
Opacity: degree of density (densest area used for assessment)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible 1
Easily discernible translucent area, details of iris siightly obscured 2*
Nacrous area, no details of iris visible, size of pupil barely discernible 3 *
Opaque cornea, iris not discernible through the opacity 4*

IRIDIC IRRITATION
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination thereof, iris still reacting to light (sluggish reaction is positive) 1 *
No reaction to light, hemorrhage, gross destruction (any or al 1 of these) 2 *

CONJUNCTIVAL IRRITATION
Redness (refers to palpebral and bulbar conjunctivae, cornea and iris).
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse, crimson color, individual vessels not easily discernible 2 *
Diffuse beefy red 3 *

Chemosis: lids and/or nictitating membranes
No swelling 0
Any swelling above normal (includes nictitating membranes) 1
Obvious swelling with partial eversion of lids 2 *
Swel 1 i ng wi th 1 i ds about hal f cl osed 3 *
Swelling with lids more than half closed 4 *

*Starred figures indicate positive effect

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

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

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

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 48 hrs

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Test item showed a primary irritation score of: 0.67, when applied to the conjunctival sac of the rabbit eye.
In the area of application, black staining of the cornea, conjunctivae, nictitating membrane, sclera and eyelashes by pigment or coloring of the test article was observed in all treated animals until termination of observation (72 hours).
No corrosion of the cornea was observed at any of the measuring intervals.

INDIVIDUAL FINDINGS,
Animal No. 87, male.
After 1 hour: Conjunctivae erythema not visible; edema generally clear visible. Sclera: not visible. Discharge: moderate. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 24 hours: Conjunctivae erythema not visible. Sclera: vessels, generally slight visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 48 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 72 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.

Animal No. 88, male.
After 1 hour: Conjunctivae erythema not visible; edema generally clear visible. Sclera: not visible. Discharge: severe. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 24 hours: Conjunctivae erythema not visible; edema, ventral slight. Nictitating membrane: slight edema. Sclera: vessels, dorsal visible. Discharge: slight. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 48 hours: Conjunctivae erythema not visible. Nictitating membrane: slight edema. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 72 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.

Animal No. 89, female.
After 1 hour: Conjunctivae erythema not visible; edema generally clear visible. Sclera: not visible. Discharge: severe. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 24 hours: Conjunctivae erythema not visible; edema, ventral slight. Nictitating membrane: slight edema. Sclera: vessels, ventral slight visible. Discharge: slight. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 48 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 72 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.

Other effects:

VIABILITY / MORTALITY AND CLINICAL SIGNS
No acute clinical symptoms were observed in the animals during the test period and no mortality occured.

BODY WEIGHTS
The body weight gain of all rabbits was similar.

Any other information on results incl. tables

Eye irritation scores for each animal

Animal N.	Reaction	1 hr	24 hrs	48 hrs	72 hrs	Mean at 24/48/72 hrs
87M	Cornea opacity	0	0	0	0	0.00
	Iris	0	0	0	0	0.00
	Conjunctivae redness	0	0	0	0	0.00
	Conjunctivae chemosis	2	0	0	0	0.00
88M	Cornea opacity	0	0	0	0	0.00
	Iris	0	0	0	0	0.00
	Conjunctivae redness	0	0	0	0	0.00
	Conjunctivae chemosis	2	1	0	0	0.33
89F	Cornea opacity	0	0	0	0	0.00
	Iris	0	0	0	0	0.00
	Conjunctivae redness	0	0	0	0	0.00
	Conjunctivae chemosis	2	1	0	0	0.33

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The substance is not an eye irritant to rabbits.

Executive summary:

The irritancy potential of the test material to the eye of New Zeland white rabbit was assessed, according to the OECD Guideline 405. The substance was applied to the right eye of three rabbits (two males and one female) and the animals were examined for signs of irritation/damage (in iris, cornea and conjuctivae) at 1, 24, 48 and 72 hours following treatment.

Black staining of the cornea, iris, conjuctivae, nictitating membrane, sclera and of the eyelashes by the test material was noted in all animals until termination of observation. No iridal or cornea irritation effects were observed. Erythema was not visible in any of the animals throught the study. Conjuctivae discharge and chemosis was observed in all animals after 1 hour and persisted after 24 hours for two out of three animals. No corossion and no mortality occured. The mean score for conjuctival chemosis was 0.33 for 2 rabbits and 0 for 1 rabbit.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for irtitis, lower than 2 for both conjunctival redness and oedema, in all animals, thus the substance can be considered as non irritating to eye, according to the CLP Regulation (EC) No 1272/2008.