[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July from 18 to 29, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

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:

Direct Blue 267

IUPAC Name:

Direct Blue 267

Specific details on test material used for the study:

For the purpose of the study the test material was ground to a powder prior to application.
The pH of the test material (10 % w/w aqueous preparation) was determined prior to commencement of the study and found to be 6.6.

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, UK.
- Age at study initiation: twelve to twenty weeks old.
- Weight at study initiation: weight range of 2.0 to 3.5 kg.
- Housing: animals were individually housed in suspended cages.
- Diet: certified rabbit diet (Code 5322) supplied by BCM IPS Limited, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: at least five days.
- Health check: immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23 °C
- Humidity: 30 to 70 %
- Air changes: at least fifteen changes per hour.
- Photoperiod: lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Test system

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml of the test material, which was found to weigh approximately 88 mg

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

Three rabbits

Details on study design:

APPLICATION
- Schedule: initially, a single rabbit was treated. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.
- Application: test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material and then released.

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by the use of 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 according to the six point scale.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation from Draize J. H. (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC pA8 to 49.

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved).
Using the numerical data obtained a modified version of the system described by Kay J Hand Calandra J C (1962), J. Soc. Cosmet. Chern. 13, 281-289 was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material.

CONJUNCTIVAE
(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal 0
Vessels definitely injected above normal 1
More diffuse, deeper crimson red, individual vessels not easily discernible 2
Diffuse beefy red 3

(B) Chemosis
No swelling 0
Any swelling above normal (includes nictitating membrane) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids half closed to completely closed 4

(C) 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 a considerable area around the eye 3

THE TOTAL SCORE = (A + B + C) × 2 MAXIMUM TOTAL = 20

IRIS
(D) Values
Normal 0
Folds above normal, congestion, swelling, circumcorneal injection (any or all 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 = D × 5

CORNEA
(E) Degree of Opacity (most dense area used)
No opacity 0
Scattered or diffuse areas, details of iris clearly visible 1
Easily discernible translucent areas, details of iris slightly obscured 2
Opalescent areas, no details of iris visible, size of pupil barely discernible 3
Opaque, iris not discernible through the opacity 4

(F) Area of Cornea Involved
One quarter (or less) but not zero 0
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 = (E × F) × 5 MAXIMUM TOTAL = 80

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

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

Reversibility:

fully reversible within: 7 days

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 8.0 and was classified as a mild irritant (class 4 on 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Black staining of the fur was noted around all treated eyes during the study. No corneal or iridial effects were noted during the study. Minimal to moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24-hour observation with minimal conjunctival irritation in all treated eyes at the 48-hour observation and in one treated eye at the 72-hour observation.
Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.

Any other information on results incl. tables

Individual scores and individual total scores for ocular irritation

Animal no.	Reaction	1 hr	24 hrs	48 hrs	72 hrs	7 days	Mean 24/48/72 hrs
Male 21	Cornea	0	0	0	0	0	0.00
Male 71	Cornea	0	0	0	0	0	0.00
Male 72	Cornea	0	0	0	0	0	0.00
Male 21	Iris	0	0	0	0	0	0.00
Male 71	Iris	0	0	0	0	0	0.00
Male 72	Iris	0	0	0	0	0	0.00
Male 21	Conjunctival redness	2	2	1	1	0	1.33
Male 71	Conjunctival redness	1	1	1	0	0	0.67
Male 72	Conjunctival redness	1	1	1	0	0	0.67
Male 21	Conjunctival oedema	2	2	1	0	0	1.00
Male 71	Conjunctival oedema	1	1	0	0	0	0.33
Male 72	Conjunctival oedema	1	0	0	0	0	0.00

	21 Male, IPR=2					71 Male, IPR=2				72 Male, IPR=2
Time after treatment	1 hrs	24 hrs	48 hrs	72 hrs	7 d	1 hrs	24 hrs	48 hrs	72 hrs	1 hrs	24 hrs	48 hrs	72 hrs
Cornea
E = degree of opacity	0	0	0	0	0	0	0	0	0	0	0	0	0
F = area of cornea involved	0	0	0	0	0	0	0	0	0	0	0	0	0
Score (E × F) × 5	0	0	0	0	0	0	0	0	0	0	0	0	0
Iris
D	0	0	0	0	0	0	0	0	0	0	0	0	0
Score (D × 5)	0	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctival
A = Redness	2	2	1	1	0	1	1	1	0	1	1	1	0
B = Chemosis	2	2	1	0	0	1	1	0	0	1	0	0	0
C = Discharge	2Sf	1Sf	0Sf	0Sf	0Sf	1Sf	1Sf	1Sf	0Sf	1Sf	1Sf	1Sf	0
Score (A + B + C) × 2	12	10	4	2	0	6	6	4	0	6	4	4	0
Total Score	12	10	4	2	0	6	6	4	0	6	4	4	0

IPR = Initial pain reaction; 2 means that rabbit blinks and tries to open eye, but reflex closes it; slight initial pain

Sf= Black staining of the fur around the treated eye

Individual total scores and group mean scores for ocular irritation

	Individual total scores at
Rabbit no.	1 hrs	24 hrs	48 hrs	72 hrs	7 days
Male 21	12	10	4	2	0
Male 71	6	6	4	0	-
Male 72	6	4	4	0	-
Group total	24	20	12	2	0
Group Mean Score	8.0	6.7	4.0	0.7	0.0

- = Observation not required

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Not eye irritant.

Executive summary:

The study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the OECD guideline 405 and the EU Method B5.

A single application of the test material to the non-irrigated eye of three rabbits produced minimal to moderate conjunctival irritation. Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.

The test material produced a maximum group mean score of 8.0 and was classified as a mild irritant (class 4 on 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Black staining of the fur was noted around all treated eyes during the study. No corneal or iridial effects were noted during the study. Minimal to moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24-hour observation with minimal conjunctival irritation in all treated eyes at the 48-hour observation and in one treated eye at the 72-hour observation.

Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.

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 of the treated animals. Alle effects were reversible within 7 days.

Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC 1272/2008).