Disodium 3,3'-[cyclohexylidenebis[(2-methyl-4,1-phenylene)azo]]bis(4,6-dihydroxynaphthalene-2-sulphonate)

Endpoint summary

Description of key information

Skin Irritation: Non irritant
Eye Irritation: Irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

other: read across from similar substacne

Adequacy of study:

key study

Study period:

May 7, 1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing procedures.

Qualifier:

according to guideline

Guideline:

other: "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).

Deviations:

not specified

GLP compliance:

no

Species:

rabbit

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Russian breed- Age at study initiation: no data- Weight at study initiation: 2.2 to 3.2 kg- Housing: They were housed singly in metal cages (overall dimensions 47x32x34 cm).- Diet: A standard diet of Nafag ad libitum.- Water: drinking water were given ad libitum.- Acclimation period: 8 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18 ± 1 °C- Humidity (%): 55 ± 5 %- Photoperiod (hrs dark / hrs light): 14 hours light/day

Type of coverage:

occlusive

Preparation of test site:

other: shaved and abraded

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 0.5 g .

Duration of treatment / exposure:

The gauze patches were removed 24 hours after application.

Observation period:

24 h, 48 h, 72 h and 4th day, 6th day, 8th day

Number of animals:

6 rabbits (3 ♂; 3 ♀)

Details on study design:

TEST SITE- % coverage: 2.5 X 2.5 cm- Type of wrap if used: The patch was covered with an impermeable foil of 5x5 cm, which was fixed to the body of the animals with adhesive tape.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: intact and abraded skin

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: intact and abraded skin

Interpretation of results:

other: not classified

Conclusions:

The substance is not irritant for skin rabbit.

Executive summary:

The substance has been tested for skin irritation according to the method inserted into the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959), the US Association of Food and Drug Officials (AFDO). Under the conditions of the present experiment the test preparation was found to cause none irritation when applied to intact and abraded rabbit skin.

The substance does not show signs of irritation according to The CLP Regulation (EC n. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

other: "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO).

GLP compliance:

no

Remarks:

pre GLP

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: russian breed.
- Weight at study initiation: 2.1 to 2.9 kg.
- Acclimation period: at least 8 days.
- Housing: animals were kept separately in metal cages (overall dimensions 47x32x34 cm).
- Diet: standard diet of Nafag, ad libitum.
- Water: drinking water, ad libitum.
- Health check: only rabbits showing normal ophtalamic findings were included in the test.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 1 °C
- Relative humidity: 55 ± 5 %
- Photoperiod: 14 hours light/day

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 g

Duration of treatment / exposure:

1 minute in three animals (eyes of the remaining 3 animals were not rinsed).

Observation period (in vivo):

8 days

Number of animals or in vitro replicates:

3 males and 3 females

Details on study design:

APPLICATION
The test substance was introduced into the conjunctival sac of the left eye with a spatula. After application, the eyelids were held open for a few seconds. The right eye was left untreated and served as a control.

REMOVAL OF TEST SUBSTANCE
The eyes of 3 animals were rinsed 1 minute after application with physiologic saline.

TOOL USED TO ASSESS SCORE: hand-slit lamp.

SCORING SYSTEM
The reactions were appraised after 1 and 6 hours; 1, 2, 3, 6 and 8 days on the basis of "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the AFDO.

CORNEA
A Opacity and degree of density (most dense area scored) .
No opacity: 0
Scattered or diffuse area, 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

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
A x B x 5; Maximum possible score = 80

IRITIS
A Values
Normal: 0
Folds above normal, congestion, swelling, circumcornea 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
A x 5; Maximum possible score = 10

CONJUNCTIVAE
A Redness (refers to lids 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 about half closed to completely closed: 4

C Discharge
No discharge: 0
Any amount different from normal (does not include small amounts observed in inner cantus 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, and considerable area around the eye: 3
(A + B + C) x 2; Maximum possible score = 20

Irritation parameter:

cornea opacity score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

1

Reversibility:

not fully reversible within: 8 days

Remarks on result:

other: rinsed eye

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

1

Reversibility:

not fully reversible within: 8 days

Remarks on result:

other: rinsed eye

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

> 2

Reversibility:

not fully reversible within: 8 days in 2 animals

Remarks on result:

other: rinsed eye

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

not fully reversible within: 8 days in two animals

Remarks on result:

other: rinsed eye

Irritant / corrosive response data:

Test item was found to cause moderate irritation: mean reaction score after 48 hours in unrinsed eyes: 43.7 and in rinsed eyes: 38.3.
The irritation consisted of conjunctival reaction, of reflective hyperemia of the iris and of corneal reaction.
There was a tendency to regress. Rinsing the eyes led to practically the same results.

Mean values from gradings at 24, 48 and 72 hours

Reaction	Animal N.	Eye	After			Mean
			24 hrs	48 hrs	72 hrs
Cornea	154	not rinsed	1	1	1	1.00
	117	not rinsed	1	1	1	1.00
	25	not rinsed	1	2	2	1.67
	104	rinsed	1	1	1	1.00
	102	rinsed	1	1	1	1.00
	133	rinsed	1	1	1	1.00
Iris	154	not rinsed	1	1	1	1.00
	117	not rinsed	1	1	1	1.00
	25	not rinsed	1	1	1	1.00
	104	rinsed	1	1	1	1.00
	102	rinsed	1	1	1	1.00
	133	rinsed	1	1	1	1.00
Conjunctival redness	154	not rinsed	3	3	3	3.00
	117	not rinsed	3	3	3	3.00
	25	not rinsed	3	3	3	3.00
	104	rinsed	3	3	3	3.00
	102	rinsed	2	3	2	2.33
	133	rinsed	3	3	1	2.33
Conjunctival chemosis	154	not rinsed	2	1	1	1.33
	117	not rinsed	2	1	1	1.33
	25	not rinsed	2	1	1	1.33
	104	rinsed	2	2	1	1.67
	102	rinsed	2	1	1	1.33
	133	rinsed	2	1	1	1.33

Rabbit eye irritation scores-feference procedure

	1 hrs							6 hrs							1 days							2 days
Rabbit No.	154	117	25	140	102	133		154	117	25	140	102	133		154	117	25	140	102	133		154	117	25	140	102	133
Eye, left/right	n	n	n	r	r	r		n	n	n	r	r	r		n	n	n	r	r	r		n	n	n	r	r	r
I . Cornea
A Opacity	0	0	0	0	0	0		0	0	0	0	0	0		1	1	1	1	1	1		1	1	2	1	1	1
B Area involved	0	0	0	0	0	0		0	0	0	0	0	0		4	4	4	4	4	4		4	4	4	4	4	4
a = AxBx5 (Max. 80)	0	0	0	0	0	0		0	0	0	0	0	0		20	20	20	20	20	20		20	20	20	20	20	20
II . Iritis	1	0	1	0	0	0		1	1	1	1	1	1		1	1	1	1	1	1		1	1	1	1	1	1
b = Score x 5 (Max. 10)	5	0	5	0	0	0		5	5	5	5	5	5		5	5	5	5	5	5		5	5	5	5	5	5
III . Conjunctivae
A Redness	2	3	3	2	1	2		3	3	3	2	1	2		3	3	3	3	2	3		3	3	3	3	3	3
B Chemosis	2	3	2	2	1	2		2	2	2	2	2	2		2	2	2	2	2	2		1	1	1	2	1	1
C Discharge	2	3	3	2	1	2		3	2	2	3	3	3		3p	2	2p	3p	2p	2		2	2	2p	3	3	1
c = (A+B+C)x2 (Max. 20)	12	18	16	12	6	12		16	14	14	14	12	14		16	14	14	16	12	14		12	12	12	16	14	10
Total = a+b+c (Max. 110)	17	18	21	12	6	12		21	19	19	19	17	19		41	39	39	41	37	39		37	37	57	41	39	35
Group mean	18.7			10.0				19.7			18.3				39.7			39.0				43.7			39.3

	3 days							4 days							8 days
Rabbit No.	154	117	25	140	102	133		154	117	25	140	102	133		154	117	25	140	102	133
Eye, left/right	n	n	n	r	r	r		n	n	n	r	r	r		n	n	n	r	r	r
I . Cornea
A Opacity	1	1	2	1	1	1		1	1	2	1	1	1		1	1	1	1	1	1
B Area involved	4	4	4	4	4	4		4	4	4	4	4	4		4	4	4	4	4	4
a = AxBx5 (Max. 80)	20	20	40	20	20	20		20	20	40	20	20	20		20	20	20	20	20	20
II . Iritis	1	1	1	1	1	1		1	1	1	1	1	1		1	1	1	1	1	1
b = Score x 5 (Max. 10)	5	5	5	5	5	5		5	5	5	5	5	5		5	5	5	5	5	5
III . Conjuctivae
A Redness	3	3	3	3	2	1		1	2	1	1	1	1		1	1	1	1	1	0
B Chemosis	1	1	1	1	1	1		1	1	1	1	0	0		1	1	1	1	1	0
C Discharge	1	1	1p	1p	1	1		1	1	1	0	1	1		1	1	1	0	1	1
c = (A+B+C)x2 (Max. 20)	10	10	10	10	8	6		6	8	6	4	4	4		6	6	6	4	6	2
Total = a+b+c (Max. 110)	35	35	55	35	33	31		35	33	51	29	29	29		31	31	31	29	31	27
Group mean	41.7			33.0				39.3			29.0				31.0			29.0

n = not rinsed

r = rinsed after 1 minute

p = purulent

Interpretation of results:

other: Irritant according to the CLP Regulation

Conclusions:

Irritant

Executive summary:

Method

The substance has been tested for eye irritation according to the method inserted into the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959), the US Association of Food and Drug Officials (AFDO). Under the conditions of this experiment 6 rabbits were tested with 0.1 g of test item inserted into the conjunctival sac of the left eye and the lids were held open for a few seconds. The right eye was not treated and served as a control. In 3 of the 6 rabbits approximately 1 minute after treatment both eyes were rinsed with physiologic saline. The reactions were appraised with a slit lamp after 1 and 6 hours; 1, 2, 3, 6 and 8 days.

Results

Under the conditions of this experiment preparation the test item was found to cause moderate irritation when applied to the rabbit eye mucosa.

Rinsing the eyes led to practically the same results. There was a tendency to regress.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

The test substance was evaluated for its skin irritation potential by one test according to the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO)".

The purity of test item was 90 % and 0.5 g of the test substance was applied on the skin of six rabbits using gauze patches, then covered by impermeable foil of 5 x 5 cm, which was fixed to the body of the animals with adhesive tape. After 24 hours the gauzes were removed and the skin observed for 8 days.

The mean score of observed values of irritation/corrosion , calculated for each rabbit, after 24, 48, 72 hours, were zero, therefore the substance is considered as non irritant.

Eye Irritation

The test substance was evaluated for its eye irritation potential according to the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO)".

The test article (0.1 g per animal) was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from 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. The eyes of the animals were observed for 8 days.

The mean score of observed values of irritation/corrosion , calculated for each rabbit, after 24, 48, 72 hours, were evaluated as follow:

rabbit N° 140: Cornea opacity: 1, Iris: 1, Conjunctivae redness: 3, Conjunctivae chemosis: 1.66

rabbit N° 102: Cornea opacity: 1, Iris: 1, Conjunctivae redness: 2.33, Conjunctivae chemosis: 1.33

rabbit N° 133: Cornea opacity: 1, Iris: 1, Conjunctivae redness: 2.33, Conjunctivae chemosis: 1.33

The high value of redness is justified because the substance is an orange dye and the colour of substance can interfere with the score of analysis.

The other values (Iris, conjunctiva chemosis and corneal opacity) are not so high to justify the classification criteria as eye damage category 1. During the observation period for conjunctiva redness and chemosis there is a slight trend to the recovery. Rinsing the eyes led to practically the same results. The observation period does not meet the criteria for the CLP Classification therfore a classification based on an expert judgement is reported.

Effects on eye irritation: irritating

Justification for classification or non-classification

SKIN IRRITATION/CORROSION

A corrosive substance is a substance that produces destruction of skin tissue, namely, visible necrosis through the epidermis and into the dermis, in at least 1 tested animal after exposure up to 4 hour duration.

Three subcategories are provided within the corrosive category 1:

Subcategory 1A where responses are noted following up to 3 minutes exposure and up to 1 hour observation;

Subcategory 1B where responses are described following exposure between 3 minutes and 1 hour and observations up to 14 days;

Subcategory 1C where responses occur after exposures between 1 hour and 4 hours and observations up to 14 days

To classify a substance as irritant, the major criterion is that at least 2 of 3 tested animals have a mean score of ≥ 2,3 - ≤ 4,0.

Category 2:

- Mean value of ≥ 2,3 - ≤ 4,0 for erythema/ eschar or for oedema in at least 2 of 3 tested animals from grading at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions; or

- Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

- In some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal but less than the criteria above.

The substance is not classified as skin irritant because in the performed tests it doesn't meet the classification criteria of the CLP regulation n. 1272/2008.

 

EYE IRRITATION

Serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

Eye irritation means the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

Irreversible effects on the eye (Category 1):

If, when applied to the eye of an animal, a substance produces:

- at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days; and/ or

- at least in 2 of 3 tested animals, a positive response of corneal opacity ≥ 3 and/or iritis > 1,5 calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight. In this context, persistent lesions are considered those which are not fully reversible within an observation period of normally 21 days. Substances are also classified in Category 1 if they fulfil the criteria of corneal opacity ≥ 3 or iritis > 1,5 detected in a Draize eye test with rabbits, recognising that such severe lesions usually do not reverse within a 21-day observation period.

The substance does not meet the requirements to be classified in Category 1.

Irritating to eyes (Category 2)

when applied to the eye of an animal, a substance produces:

- at least in 2 of 3 tested animals, a positive response of: corneal opacity ≥ 1 and/or iritis ≥ 1, and/or conjunctival redness ≥ 2 and/or conjunctival oedema (chemosis) ≥ 2

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

The substance is not fully reversible within observation period of 8 days. The observation period does not meet the criteria for the CLP Classification therfore the classification is based on expert judgement: the dermal effects are expected to be reversible within 21 days therefore the substance is classified as irritating to eyes (Category 2).