2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-, coupled with 3-aminophenol, diazotized 5-amino-2-[(4-aminophenyl)amino]benzenesulfonic acid and diazotized benzenamine, sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November from 15 to 22, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: readings was difficult and in some cases not feasible, due to substance staining

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

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

IUPAC Name:

Direct Black 168

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: PI PETIT (El Masnou, Barcelona, Spain).
- Age at study initiation: approximately 14 weeks.
- Weight at study initiation: 2.3 - 2.5 kg.
- Housing: housed individually in stainless steel cages (48 x 59 x 37 cm), with a grille floor, placed on racks.
- Diet: standard rabbit diet UAR 112, ad libitum.
- Water: an automatic drinking system, ad libitum.
- Acclimation period: at least 7 days.
- Health check: both eyes of each animal were examined before treatment in order to reject any animal showing eye alterations.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 ± 2 °C, sometimes reaching 16 and 22 °C.
- Humidity: 40 - 70 %, sometimes reaching levels of 28 - 39 % and 75 - 80 %.
- Photoperiod: 12 hour light/12 hour dark.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

- Amount applied: 0.1 ml
- Application: the test substance was instilled, undiluted, in a single application.

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3 males

Details on study design:

APPLICATION OF TEST MATERIAL
The animal was immobilized and the product was poured into the right conjunctival sac, made perfectly accessible by care fully pulling out the lower lid. After application of the test substance, the lids of the treated eye were held closed for approximately one second.

REMOVAL OF TEST SUBSTANCE
- Washing: the treated eye was washed with 0.9 % w/v sodium chloride solution, after Colirusi Fluoresceina application.
- Time after start of exposure: 24 hours.

TOOL USED TO ASSESS SCORE
Followving the reading carried out 24 hours after treatment, Colirusi Fluoresceina was applied to the treated eye of each animal.

SCORING SYSTEM
The degree of eye irritation was evaluated for each rabbit 1, 24, 48 and 72 hours after treatment, following the guideline.
All the measures carried out at each 24, 48 and 7 hour examination for each type of lesion are used respective mean values.

- Corneal Irritation
Opacity: degree of density (densest area used for assessment)
No ulceration or opacity............................................................................................................................................... 0
Scattered or diffuse areas of opacity (apart from slight loss of normal luster), details of iris clearly visible............. 1
Easily discernible translucent area, details of iris slightly blurred................................................................................2
Opalescent area, details of iris not visible, countour of pupil barely discernible.........................................................3
Opaque cornea, iris not discernible through the opacity............................................................................................. 4

- The area of corneal opacity was recorded according to the following scale:
A quarter or less, but not zero ......................1
More than a quarter, less than half................2
More than half, less than three quarters........3
More than three quarters, up to whole area...4

- Iridic Irritation
Normal............................................................................................................................................................................0
Deeply marked folds, congestion, swelling, moderate hyperemia around corena or injection, one or more
of the aforementioned, iris conserving reaction to light................................................................................................ 1
No reaction to light, hemorrhage, serious damage (any or all of the above)................................................................ 2

- Conjuctival Irritation
Redness (refers to palpebral and bulbar conjunctivae, cornea and iris).
Normal blood vessels................................................................................................................................................... 0
Some clearly hyperemic blood vessels (injected)........................................................................................................ 1
Diffuse, crimson color, individual vessels difficult to discern...................................................................................... 2
Diffuse deep red........................................................................................................................................................... 3

- Edema: lids and/or nictitating membranes
No swelling.................................................................................................................................................................... 0
Some swelling above normal (includes nictitating membranes).....................................................................................1
Obvious swelling with partial eversion of lids............................................................................................................... 2
Swelling wi th lids about half closed............................................................................................................................. 3
Swelling with lids more than half closed....................................................................................................................... 4

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

1

Reversibility:

not fully reversible within: 7 days, in one animal

Results 2

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Remarks on result:

not determinable

Remarks:

due to the coloring from test substance

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal: 1/3

Time point:

24/48/72 h

Score:

2.33

Reversibility:

not fully reversible within: 7 days

Remarks on result:

not determinable

Remarks:

in 2 out of 3 animals

Results 4

Irritation parameter:

chemosis score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

not fully reversible within: 7 days, in one animal

Remarks on result:

other: in the 3rd animal reaction were not recorded within 7 days

Irritant / corrosive response data:

In the course of the first 24 hours after administration, all the animals showed edernatic lesions ranging from swelling of the lids (grade 1) to more than half closed lids (grade 4).
Discharge was also observed ranging from light to moistening of the lids and surrounding hair, affecting a considerable area around the eye and injected vessels in the bulbar conjunctivae.
24 hours following administration, all the animals showed diffuse opacity of the cornea (grade 1) ranging from an affected area of one quarter (grade 1) to near totality of the cornea (grade 4). Due to the colouring produced by the test substance in the eyes of the animals, diffused crimson vessels (grade 3) were observed in the palpebral conjunctivae of only one animal and none of the animal's irises could be clearly observed .
2 , 3 and 4 days following administration, observed ocular injuries were seen to be subsiding.
7 days post-administration, one animal showed vessels of the palpebral conjunctivae clearly more injected than normal (grade 1). Also, another animal showed nearly half closed, swollen lids (grade 3) and diffuse opacity of the cornea (grade 1) which occupied less than one quarter of the total area (grade 1).

Other effects:

CLINICAL SIGNS
The behaviour and physical condition of the rabbits were normal throughout the Study.

Any other information on results incl. tables

The eye irritation scores for each animal are presented in the table below.

Animal N.	Reaciton		1 h	24 hrs	48 hrs	72 hrs	4 d	7 d	Mean 24/48/72 hrs
3989	Cornea	Density	-	1	1	1	1	0	1.00
		Area	-	4	3	2	1	0	-
	Iris		-	-	-	-	-	-	-
	Conjunctiva	Hyperemia	-	-	-	-	-	-	-
		Oedema	3	1	1	1	1	0	0.33
4101	Cornea	Density	-	1	0	0	0	0	0.33
		Area	-	1	0	0	0	0	-
	Iris		-	-	-	-	0	0	-
	Conjunctiva	Hyperemia	-	3	2	2	2	1	2.33
		Oedema	2	2	1	1	1	0	1.33
3993	Cornea	Density	-	1	1	1	1	1	1.00
		Area	-	4	3	2	2	1	-
	Iris		-	-	-	-	-	-	-
	Conjunctiva	Hyperemia	-	-	-	-	-	-	-
		Oedema	3	4	4	4	4	3	4.00

- : non quantifiable due to the colouring from test substance.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The substance is considered to be an eye irritant to rabbits.

Executive summary:

The substance was assessed for eye irritation potential to rabbits, according to the OECD guideline 405. The right eye of three male rabbits was treated with the substance (non diluted) and after 24 hours the eyes were washed.

In the course of the first 24 hours after administration, the substance induced edematic lesions, ranging from some swelling above normal of the lids (grade 1) to more than halfclosed lids (grade 4). Also observed were injected vessels in the bulbar conjunctivae of all animals and discharge ranging from mild to moistening of the lids and surrounding hair, affecting a considerable area around the eye. All the animals showed opacity of the cornea (grade 1) ranging from an affected area of one quarter (grade 1) to near totality of the cornea (grade 4). Due to the colouring produced by the test substance in the palpebral conjunctivae and iris of some of the animals, diffused crimson vessels (grade 3) were observed in only one animal. 2, 3 and 4 days following administration, observed ocular injuries had begun to subside.

In the reading carried out 7 days post-administration, one animal showed vessels clearly more injected than normal (grade 1) in the palpebral conjunctivae. Also, another animal showed nearly half closed, swollen lids (grade 3) and opacity of the cornea (grade 1) which occupied less than one quarter of the total area (grade 1).

Conclusion

The substance is considered to be an eye irritant to rabbits.