Sodium 6-amino-5-[[4-chloro-3-[[(2,4-dimethylphenyl)amino]sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation, other

Remarks:

in vivo

Type of information:

other: read acorss from suporting substance (analogue structure or surrogate)

Adequacy of study:

key study

Study period:

June from 14th to 29th, 1993

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:

1993

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 1, AR336

IUPAC Name:

similar substance 1, AR336

Specific details on test material used for the study:

The test material was used as supplied.

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: approximately 12 to 16 weeks old.
- Weight at study initiation: 2.74 - 2.90 kg.
- Housing: animals were individually housed in suspended metal cages.
- Diet: mains food RABMA Rabbit Diet, ad libitum.
- Water: mains drinking water, ad libitum.
- Acclimation period: minimum 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. Animals showing evidence of ocular Iesions were rejected and replaced.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 23 °C
- Humidity: 56 - 66 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: 12 hours light and 12 hours darkness.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml of the test material, which was found to weigh approximately 65 mg

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

Two males and one female

Details on study design:

PROCEDURE
- Proceeding: one rabbit was initially 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 coniunctival 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 irmediately after application, to prevent loss of the test material, and then released.
- Pain prevention: in order to minimise pain on application of the test material, one drop of local anaesthetic ("Ophthaine", 0.5 % proxymetacaine hydrochloride) was instilled into both eyes of animals 1 - 2 minutes before treatment.

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by use of the light source from a standard ophthalmoscope. Confirmation of corneal opacity was made by using sodium fluorescein B.P., if required.

SCORING SYSTEM
Immediately after administration of the test material, an assessment of the initial pain reaction was made.
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. 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 ocular effects were also noted.
Additional observations were made on days seven and fourteen to assess the reversibility of the ocular effects.

CONJUNCTIVAE
(A) Redness (refers to pelpebral 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) Chemsis
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 comèpletely 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
Diacharge with moinstening of the lids and hairs just adjacent to lids 2
Diacharge with moinstening of the lids and hairs a considerable area around the eye 3

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

(D) IRIS
Normal 0
Folds above normal, congestion, swelling, circumcorneal injection (any or all of these combination of any thereof) iris still reacting to light (slugging reaction is positive) 1

TOTAL SCORE (D) x 5
MAXIMUM TOTAL: 10

CORNEA
(E) Degree of opacity (most degree 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 invisible 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 half but less than three quarters 3
Greater than three quarters, up to whole area 4

TOTAL SCORE (E x F) x 5
MAXIMUM TOTAL: 80

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: 14 days

Results 2

Irritation parameter:

iris score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 7 days

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

>= 2

Reversibility:

fully reversible within: 14 days

Results 4

Irritation parameter:

chemosis score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

Residual test material was noted around the treated eye of all animals throughout the study.
Red-coloured staining was noted in all treated eyes one hour after treatment.

Areas of diffuse corneal opacity were noted in all treated eyes one hour after treatment, at the 24, 48 and 72-hour observations and in one treated eye at the 7-day observation. Corneal opacity was confirmed using sodium fluorescein B.P. in all treated eyes one hour after treatment. Vascularisation of the cornea was noted in one treated eye at the 7-day observation.
Red-coloured staining prevented accurate evaluation of the iris in all treated eyes one hour after treatment. Iridial inflammation was noted in all treated eyes at the 24-hour observation and in two treated eyes at the 48 and 72-hour observations. No other iridial effects were noted.

Red-coloured staining prevented accurate evaluation of conjunctival redness in all treated eyes one hour after treatment slight conjunctival chemosis and severe discharge were noted in all treated eyes at this time. Moderate conjunctival irritation was noted in all treated eyes at the 24-hour observation with minimal to moderate conjunctival irritation at the 48-hour observation. Minimal coniunctival irritation was noted in two treated eyes at the 72-hour observation. The reaction increased to severe conjunctival irritation in one treated eye at the 72-hour observation with minimal coniunctival irritation at the 7-day observation.
No ocular effects were noted in two treated eyes seven days after treatment and in one treated eye fourteen days after treatment.

Any other information on results incl. tables

OVERALL SCORE

Animal	Reaction	Time After Treatment						Mean 24/48/72 hrs
		1 hr	24 hrs	48 hrs	72 hrs	7 dy	14 dy
Female n. 100	Cornea	1FS	1	1	1	0	0	1.00
	Iris	?s	1	1	1	0	0	1.00
	Conjunctivae redness	?s	2	2	2	0	0	2.00
	Conjunctivae chemosis	2	2	2	1	0	0	1.67
Male n. 110	Cornea	1FS	1	1	1	1V	0	1.00
	Iris	?s	1	1	1	0	0	1.00
	Conjunctivae redness	?s	2	2	3	1	0	2.33
	Conjunctivae chemosis	2	2	2	2	1	0	2.00
Female n. 113	Cornea	1FS	1	1	1	0	0	1.00
	Iris	?s	1	0	0	0	0	0.33
	Conjunctivae redness	?s	2	2	1	0	0	1.67
	Conjunctivae chemosis	2	2	1	1	0	0	1.33

INDIVIDUAL SCORES AND INDIVIDUAL TOTAL SCORES FOR OCULAR IRRITATION

Famale N. 100

Time After Treatment	1 hr	24 hrs	48 hrs	72 hrs	7 dy	14 dy
Cornea
E: degree of Opacity	1FS	1	1	1	0	0
F: area of Opacity	3	3	2	2	0	0
Score (E x F) x 5	15	15	10	10	0	0
Iris
D	?s	1	1	1	0	0
Score (D x 5)	0-10	5	5	5	0	0
Conjunctivae
A: redness	?s	2	2	2	0	0
E: chemosis	2	2	2	1	0	0
E: discharge	3Re	2Re	0Re	0Re	0Re	0Re
Score (A + B + C) x 2	10-16	12	8	6	0	0
Total score	25-41	32	23	21	0	0

F: due to red-coloured staining of the cornea, sodium fluorescein B.P. was used to aid confirmation of corneal opacity

S: red-coloured staining

Re: residual test material around treated eye

?s: red-coloured staining preventing accurate evaluation of ocular effect

V: vascularisation of the cornea

Male N. 110

Time After Treatment	1 hr	24 hrs	48 hrs	72 hrs	7 dy	14 dy
Cornea
E: degree of Opacity	1FS	1	1	1	1V	0
F: area of Opacity	4	4	3	3	1	0
Score (E x F) x 5	20	20	15	15	5	0
Iris
D	?s	1	1	1	0	0
Score (D x 5)	0-10	5	5	5	0	0
Conjunctivae
A: redness	?s	2	2	3	1	0
E: chemosis	2	2	2	2	1	0
E: discharge	3Re	3Re	2Re	3Re	0Re	0Re
Score (A + B + C) x 2	10-16	14	12	16	4	0
Total score	30-46	39	32	36	9	0

F: due to red-coloured staining of the cornea, sodium fluorescein B.P. was used to aid confirmation of corneal opacity

S: red-coloured staining

Re: residual test material around treated eye

?s: red-coloured staining preventing accurate evaluation of ocular effect

V: vascularisation of the cornea

Male N. 113

Time After Treatment	1 hr	24 hrs	48 hrs	72 hrs	7 dy	14 dy
Cornea
E: degree of Opacity	1FS	1	1	1	0	0
F: area of Opacity	3	3	2	1	0	0
Score (E x F) x 5	15	15	10	5	0	0
Iris
D	?s	1	0	0	0	0
Score (D x 5)	0-10	5	0	0	0	0
Conjunctivae
A: redness	?s	2	2	1	0	0
E: chemosis	2	2	1	1	0	0
E: discharge	3Re	2Re	0Re	0Re	0Re	0Re
Score (A + B + C) x 2	10-16	12	6	4	0	0
Total score	25 -41	32	16	9	0	0

F: due to red-coloured staining of the cornea, sodium fluorescein B.P. was used to aid confirmation of corneal opacity

S: red-coloured staining

Re: residual test material around treated eye

?s: red-coloured staining preventing accurate evaluation of ocular effect

V: vascularisation of the cornea

Applicant's summary and conclusion

Interpretation of results:

other: Eye. Irrit. 2 (H319), according to the CLP Regulation (EC 1272/2008)

Conclusions:

Moderate 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 used followed that described in the OECD Guidelines 405. A single application of the test material to the non-irrigated eye of three rabbits produced areas of diffuse corneal opacity, iridial inflammation and moderate to severe conjunctival irritation.

Vascularisation of the cornea was confined to one treated eye. No ocular effects were noted seven or fourteen days after treatment.

The test material produced a maximum group mean score in the range of 34.3 - 42.7 and was classified as a moderate irritant (class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were equal to 1 for corneal opacity and iritis in all rabbits, equal/higher than 2 for conjunctival redness in two out of three rabbits and lower than 2 for chemosis in two out of three rabbits; all the reactions recorded were fully reversible within 14 days.

The substance can be considered as irritating to eye and a classification in category 2 (Eye. Irrit. 2 (H319) of the CLP Regulation (EC 1272/2008) can be considered as appropriated.