Reaction mass of 3,3'-(1E,1'E)-(4,4'-(6-(bis(2-hydroxyethyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl)bis(3-methoxy-4,1-phenylene))bis(diazene-2,1-diyl)dibenzenesulfonic acid and disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

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:

key study

Study period:

From May 15 to 26, 2006

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:

Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

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:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

not specified

Specific details on test material used for the study:

Test material was used as supplied.
pH of test material was determined before starting the study and found to be 9.1 at 10 % w/w aqueous preparation of test material.

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: 2.0 to 3.5 kg
- Housing: the animals were individually housed in suspended cages.
- Diet: Certifred Rabbit Diet (Code 5322) supplied by BCM IPS Limited, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: at least 5 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 - 23 °C
- Humidity: 30 - 70 %
- Air changes: the rate of air exchange was at least fifteen changes per hour.
- Photoperiod: the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A volume of 0.1 ml of test material, which was found to weigh approximately 87 mg was placed into the conjunctival sac of the right eye.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 rabbits.

Details on study design:

TESTING PROCEDURES
Initially, a single rabbit was treated. Test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. Upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of test material and then released.
Immediately after administration of test material, an assessment of initial pain reaction was made.
After consideration of ocular responses produced in the first treated animal, two additional animals were treated.

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

SCORING SYSTEM
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given by Draize J. H. (1977).
Any other ocular effects were also noted.
Numerical values corresponding to each animal, tissue and observation time were recorded.
Data relating to conjunctivae were designated by letter A (redness), B (chemosis) and C (discharge), those relating to iris by letter D and those relating to cornea by letters E (degree of opacity) and F (area of cornea involved). For each tissue, the score was calculated.
Using numerical data obtained, a modified version of the system described by Kay JH and Calandra JC (1962), J. Soc. Cosmet. Chem. 13, 281-289 was used to classify the ocular irritancy potential of test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. Group means of total scores for each observation were calculated. The highest of these group means (maximum group mean score) together with the persistence of reactions enabled classification of eye irritancy potential of test material.
If evidence of irreversible ocular damage is noted, test material is classified as corrosive to eye.

Draize scale for scoring ocular irritation
CONJUNCTIVAE
(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal 0
Vessels definitely injected above normal 1
More diffirse, deeper crimson red, individual vessels not easily discernible 2
Diffiuse 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 a¡ea a¡ound the eye 3

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

IRIS
(D) Values
Normal 0
Folds above normal, congestion, swelling, circumcomeal 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 x 5
MAXIMIUM TOTAL = 10

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

(F) Area of Cornea Involved
One quarter (or less) but not zero 1
Greater than one quarter but less than half 2
Greater than halfbut less than three quarters 3
Greater than three quarters, up to whole area 4

THE TOTAL SCORE = (E x F) x 5
MAXIMIUM 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: 72 hrs

Results 4

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 48 hrs

Irritant / corrosive response data:

Yellow-coloured staining of the fur was noted around all treated eyes throughout the study. No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one hour after treatment.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal to moderate conjunctival irritation noted in all treated eyes at the 24-hours observation and minimal conjunctival irritation noted in all treated eyes at the 48-hours observation.
All treated eyes appeared normal at the 72-hours observation.
Test material produced a maximum group mean score of 13.7 and was classified as a mild irritant to the rabbit eye according to a modified Kay and Calandra classification system.

Any other information on results incl. tables

Animal no.	Reaction	Observation at				Mean 24/48/72 hrs
		1 hr	24 hrs	48 hrs	72 hrs
64 Male	Corneal opacity	0	0	0	0	0.00
12 Male	Corneal opacity	0	0	0	0	0.00
14 Male	Corneal opacity	0	0	0	0	0.00
64 Male	Iris	1	0	0	0	0.00
12 Male	Iris	0	0	0	0	0.00
14 Male	Iris	0	0	0	0	0.00
64 Male	Conjunctival redness	2	2	1	0	1.00
12 Male	Conjunctival redness	2	1	1	0	0.67
14 Male	Conjunctival redness	2	1	1	0	0.67
64 Male	Conjunctival chemosis	2	1	0	0	0.33
12 Male	Conjunctival chemosis	2	1	0	0	0.33
14 Male	Conjunctival chemosis	2	1	0	0	0.33

Individual scores and individual total scores for ocular irritation

	64 Male				12 Male				14 Male
	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
E: Degree of Opacity	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
Score (E × F) × 5	0	0	0	0	0	0	0	0	0	0	0	0
IRIS
D	1	0	0	0	0	0	0	0	1	0	0	0
Score (D × 5)	5	0	0	0	0	0	0	0	5	0	0	0
CONJUNCTIVAE
A: Redness	2	2	1	0	2	1	1	0	2	1	1	0
B = Chemosis	2	1	0	0	2	1	0	0	2	1	0	0
C: Discharge	2Sf	1Sf	0Sf	0Sf	2Sf	1Sf	0Sf	0Sf	2Sf	1Sf	0Sf	0Sf
Score (A + B + C) × 2	12	8	2	0	12	6	2	0	12	6	2	0
total score	17	8	2	0	12	6	2	0	12	6	2	0

Sf = yellow-coloured staining of fur around treated eve

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Not irritant.

Executive summary:

The assessment of the eye irritancy potential of test substance was done according to OECD guideline 405.

Upon a single application of test material to the non-irrigated eye of three rabbits, effects were scored 1, 24, 48 and 72 h after application. No effects on iris and cornea were noted. Slight effects on conjunctivae in terms of redness and chemosis were noted in 3/3 rabbits; such effects were completely reversible within 72 h.