5',5'''-[ethylenebis(p-phenyleneazo)]bis[1',2'-dihydro-6'-hydroxy-4'-methyl-2'-oxo-1,3'-bipyridinium] dilactate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Remarks:

source of read-across

Adequacy of study:

key study

Study period:

From 2nd to 6th January, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01

IUPAC Name:

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: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: at the start of the study the animals were approximately twelve to sixteen weeks old.
- Weight at study initiation: at the start of the study the animals weighed 2.90 - 3.34 kg.
- Housing: individually housed in suspended metal cages.
- Diet: SpilIers Rabbit Diet, Dalgety Agriculture Ltd., Almondsbury, Bristol, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: minimum acclimatisation period of five days.
- Health: immediately before the start of the test, both eyes of the three provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Animals showing evidence of ocular lesions were rejected and replaced.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 19 °C
- Humidity: 48 - 60 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The test material was used as supplied.
0.1 ml of the test material, which was found to weigh approximately 66 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid arway from the eyeball. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three rabbits

Details on study design:

PROCEDURE
One rabbit was initially treated. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by use the light source from a standard ophthalmoscope.

SCORING SYSTEM
Immediately after administration of the test material, an assessment of the initial pain reaction was made. Assessment of ocular damage/irritation rras made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation proposed by Draize J.H. 1959, Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics".
Any other adverse ocular effects were also noted.

Grades of ocular lesions
A. 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
Opalescent area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 4

B. Area of cornea involved
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

The total score = (A x B) x 5
Maximum total = 80

C. IRIS
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive) 1
No reaction to light, haemorrhage, gross destruction (any or all of these) 2

The total score = C x 5
Maximum total = 10

CONJUNCTIVAE
D. Redness: (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Blood vessels normal 0
Some blood vessels definitely hyperaemic (injected) 1
Diffuse, crimson colour, individual vessels not easily discernible 2
Diffuse beefy red 3

E. Chemosis
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
Swelling with lids about half closed 3
Swelling with lids more than half closed 4

F. Discharge
No discharge 0
Any anount different from nornal (does not include small anounts observed in inner canthus of nornal animals) 1
Di scharge with moistening of the lids and hairs just adjacent to lids 2
Di scharge with moistening of the lids and hairs a considerable area around the eye 3

The total score = (D + E + F) x 2
Maximum total = 20

Maximum total score possible = 110

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 within: 72 hrs

Results 2

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Reversibility:

fully reversible within: 72 hrs

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 72 hrs

Results 4

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 72 hrs

Irritant / corrosive response data:

The test material produced a maximum group mean score of 16.3 and was classified as a mild irritant to the rabbit eye, according to a modified Kay and Calandra classification system.
A dulling of the normal lustre of the corneal surface was noted in one treated eye one hour after treatment with diffuse corneal opacity at the 24 and 48-hour observations. No other adverse corneal effects were noted.
Iridial inflammation was noted in all treated eyes one hour after treatment and in one treated eye at the 24 and 48-hour observations. No other adverse iridial effects were noted.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and in one treated eye at the 24-hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. Residual test material was noted around the treated eye of all animals during the study.
All treated eyes appeared normal 72 hours after treatment.

Any other information on results incl. tables

Individual mean 24, 48 and 72 hours score

Skin reaction	244 female				239 female				265 female
	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs
Cornea opacity	d	1	1	0	0	0	0	0	0	0	0	0
Mean 24, 48, 72 hrs	0.7				0.0				0.0
Iris	1	1	1	0	1	0	0	0	1	0	0	0
Mean 24, 48, 72 hrs	0.7				0.0				0.0
Redness	2	2	1	0	2	1	0	0	2	1	0	0
Mean 24, 48, 72 hrs	1.0				0.3				0.0
Chemosis	2	2	1	0	1	0	0	0	1	0	0	0
Mean 24, 48, 72 hrs	1.0				0.3				0.0

Individual scores and individual total scores for ocular irritation

Skin reaction	244 female				239 female				265 female
	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs
A. Cornea opacity	d	1	1	0	0	0	0	0	0	0	0	0
B. Area involved	4	2	1	0	0	0	0	0	0	0	0	0
Score (A x B) x 5	0	10	5	0	0	0	0	0	0	0	0	0
C. Iris	1	1	1	0	1	0	0	0	1	0	0	0
Score C x 5	5	5	5	0	5	0	0	0	5	0	0	0
D. Redness	2	2	1	0	2	1	0	0	2	1	0	0
E. Chemosis	2	2	1	0	1	0	0	0	1	0	0	0
F. Discharge	3Re	2Re	1Re	0Re	2Re	0Re	0Re	0Re	2Re	0Re	0Re	0Re
Score (D + E + F) x 2	14	12	6	0	10	2	0	0	10	2	0	0
Total score	19	27	16	0	15	2	0	0	15	2	0	0

d: dulling of the normal lustre of the corneal surface

Re: residual test material around the eye

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC1272/2008)

Conclusions:

Not irritant.

Executive summary:

A study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method used followed that described in the OECD guideline 405. A single application of the test material to the non-irrigated eye of three rabbits produced iridial inflammation and moderate coniunctival irritation. Diffuse corneal opacity was confined to one treated eye. All treated eyes appeared normal 72 hours after treatment. The test material produced a maximum group mean score of 16.3 and was classified as a mild irritant to the rabbit eye according to a modified Kay and Calandra classification system. A dulling of the normal lustre of the corneal surface was noted in one treated eye one hour after treatment with diffuse corneal opacity at the 24 and 48-hour observations. No other adverse corneal effects were noted. Iridial inflammation was noted in all treated eyes one hour after treatment and in one treated eye at the 24 and 48-hour observations. No other adverse iridial effects were noted. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and in one treated eye at the 24-hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. Residual test material was noted around the treated eye of all animals during the study. All treated eyes appeared normal 72 hours after treatment.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all the three tested animals. Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC 1272/2008).