Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 19, 1994 - May 26, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

EC Number:

418-220-4

EC Name:

Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

Molecular formula:

C52H32CrN18Na3O20S4

IUPAC Name:

chromium(3+) trisodium bis(6-amino-2-{4-[2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]benzenesulfonamido}-5-[2-(3-nitro-2-oxido-5-sulfonatophenyl)diazen-1-yl]pyrimidin-4-olate)

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

rabbit

Strain:

other: Zew Zeland rabbits

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Animals: rabbit, Chbb: NZW (SPF)
- Source: Dr. Karl Thomae GmbH Postfach 1755 D-88397 Biberach an der Riss.
- Age at start of treatment: males at 12 weeks, females at 11 weeks.
- Body weight at start of acclimatization: male at 2.7 kg, females at 2.1.
- Body weight at start of treatment: male at 2.8 kg, females at 2.2 – 2.3 kg.
- Identification: by unique cage number and corresponding tags.
- Accomodation: individually in stainless steel cages with an automatic cleaning system equipped with feed hoppers, drinking water bowls and wood for gnawing.
- Diet: pelleted standard Kliba 341, Batch 83/94 rabbit maintenance diet ("Kllba", Klingentalmueble AG, CH-4303 Kaiseraugst), ad libitum.
- Water: community tap water from Itingen, ad libitum; A bacteriological, chimical and contaminant analyses were performed.
- Acclimatization: four days under test conditions after health examination. Only animals without any visual signs of illness were used for the study. The eyes of the animals were examined for intactness 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 ACH
- Photoperiod: 12 hours artificial fluorescent light (approx 100 Lux) / 12 hours dark, misic during light period.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

- Amount applied: single dose at 0.1 g/animal (left eye)

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

1 male and 2 females

Details on study design:

TREATMENT
On test day 1, 0.1 g of the test article (undiluted) was placed in the conjunctival sac of the left eye of each animal after gently pulling the lowerlid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test article. The right eye remained untreated and served as the reference control.

TOOL USED TO ASSESS SCORE: 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.

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

IRIDIC IRRITATION
Normal 0
Markedly deepened rugae, congestión, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination thereof, iris still reacting to llght (sluggish reaction is positivo) 1*
No reaction to light, hemorrhage, gross destructlon (any or all of these) 2*
Maximum scores: 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*
Maximum scores: 3

Chemosis: lids and/or nictitating membranes
No swelling 0
Any swelling above normal (includes nictitating membranes) 1
Obvious swelling with partial eversión of lids 2*
Swelling with lids about half closed 3*
Swelling with lids more than half closed 4*
Maximum scores: 4

*Starred figures indícate positive effect

OTHER OBSERVATION
Viability, mortality and clinical signs: daily during observation period.
Body weights: at the start of acclimatization, on day 1 of test (application day) and at termination of observation.

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

Reversibility:

fully reversible

Results 2

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Reversibility:

fully reversible

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible

Results 4

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible

Irritant / corrosive response data:

Irritation
Primary irritation score of: 1.67, when applied to the conjunctival sac of the rabbit eye.

Coloration
In the area of application red staining of the conjunctivae and nictitating membrane by the test article was observed.

Corrosion
No corrosion of the cornea was observed at any of the reading items.

Other effects:

Clinical signs and mortality
No systemic symptoms were observed in the animals during the test and observation period, and no mortality occurred.

Body weights
The body weight gain of all rabbits was within the normal range of variability.

Any other information on results incl. tables

Individual mean score at 24, 48 and 72 hours

Reaction	Time	Animal/sex
		4 M	5 F	6 F
Cornea	1 hr	0	0	0
	24 hrs	3*	0	0
	48 hrs	0	0	0
	72 hrs	0	0	0
Mean 24, 48 and 72 hrs		1	0	0
Iris	1 hr	0	0	0
	24 hrs	1*	0	0
	48 hrs	0	0	0
	72 hrs	0	0	0
Mean 24, 48 and 72 hrs		0.33	0	0
Conjunctivae redness	1 hr	-	-	-
	24 hrs	3*	1	0
	48 hrs	1	0	0
	72 hrs	0	0	0
Mean 24, 48 and 72 hrs		1.33	0.33	0
Conjuntivae chemosis	1 hr	2*	1	1
	24 hrs	4*	1	0
	48 hrs	1	0	0
	72 hrs	0	0	0
Mean 24, 48 and 72 hrs		1.67	0.33	0

Eye irritation score

Time period	Sex	Cornea opacity	Iris	Conjunctivae		Cumulative score	Mean cumulative score
				Redness	Chemosis
1 h	M	0	0	-	2	2	1.33
	F	0	0	-	1	1
	F	0	0	-	1	1
24 h	M	3	1	3	4	11	4.33
	F	0	0	1	1	2
	F	0	0	0	0	0
48 h	M	0	0	1	1	2	0.67
	F	0	0	0	0	0
	F	0	0	0	0	0
72 h	M	0	0	0	0	0	0.00
	F	0	0	0	0	0
	F	0	0	0	0	0

"-" could not be assessed

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The substance was found to be as non-irritant to the eye of rabbits under the test conditions.

Executive summary:

The eye irritation potential of the test substance was investigated by instillation of 0.1 g into one eye of each of three young adult New Zealand rabbits. The treated eyes were not rinsed after substance application. Scoring of irritation effects were performed 1, 24, 48 and 72 hours after test article application.

The scores of each animal at the following reading times (24, 48 and 72 hours) were used in calculating the respective mean values for each type of lesion.

The cornea and iris mean scores24/48/72 h were 0 in all animals except for animal #1 (1 for cornea and 0.33 for iris). The mean values 24/48/72 h for conjunctivae were for redness 1.33, 0.33, 0 and for chemosis 1.67,0.33, 0 in animals #1, #2 and #3 respectively. In the area of application red staining of the conjunctivae and nictitating membrane by the test article was observed. Colouration and all the other effects observed were fully reversible within the 7-day study period.