Reaction mass of sodium 3-[[4-[(4-ethoxy-m-tolyl)azo]-1-naphthyl]azo]benzenesulphonate and sodium 3-[[4-[(4-ethoxyphenyl)azo]-1-naphthyl]azo]benzenesulphonate

Endpoint summary

Description of key information

Not classified as skin irritating, according to the CLP Regulation (EC) No 1272/2008

Not classified as eye irritating, according to the CLP Regulation (EC) No 1272/2008

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The read across approach is detailed into the document attached into the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Principles of method if other than guideline:

The test was carried out according to the method given in the "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11 (February 1965).

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 - 13 weeks.
- Weight at study initiation: average 2.05 kg males and 2.22 kg females.
- Housing: rabbits were caged singly.
- Diet: commercial irradiated diet (Styles-Oxoid), ad libitum.
- Water: sterile filtered water, ad libitum.
- Acclimation period: one week prior to the start of the trial.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 1 °C
- Relative humidity: 50 - 70 %
- Photoperiod: animals were exposed to artificial light for 10 hours daily from 08.00 - 18.00 hours.

Type of coverage:

occlusive

Preparation of test site:

other: shaved and abraded

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Amount: 0.5 g
Fromulaiton: 10 g.of the test compound was mixed with 10 ml of water to make a solution of 15 ml, 0.75 ml of which was applied to each test site on a 2.5 cm square gauze pad.

Duration of treatment / exposure:

24 hours

Observation period:

72 hours

Number of animals:

3 males and 3 females

Details on study design:

TEST SITE
- Area of exposure: 24 hours prior to the dermal application, the backs of the rabbits were shaved. Two test sites lateral to the mid line of the back were used on each rabbit. Immediately before the application of the test compound, the right hand site was abraded uith the point of a sterile hypodermic needle. The abrasions were sufficiently deep to penetrate the stratum corneum but not to damage the dermis.
- % coverage: at least 10 % of the total body surface.
- Type of wrap if used: animals were covered with aluminium foil secured with "Sleek" adhesive tape. The test sites were then enclosed by a 6 wide "Coban" self adhesive bandage the edges of which were fixed to the skin by strips of "Sleek" in order to retain the test substance in close contact with the skin.

OBSERVATION TIME POINTS
After 24 hours the dressing was removed and the intact and abraded application sites were assessed for oedema and erythema. A further assessment wes made at 72 hours.

SCORING SYSTEM

Erythema and Eschar Formation
No erythema 0
Slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet red) to slight eschar formation 4

Oedema Formation
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well defined by definite raising) 2
Moderate oedema (raised approximately 1 mm) 3
Severe-oedema (raised more than 1 mm and extending beyond the area of exposure) 4

Irritation parameter:

erythema score

Basis:

animal: 6/6

Remarks:

both intact and abraded

Time point:

24/48/72 h

Score:

< 2.3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal: 6/6

Remarks:

both intact and abraded

Time point:

24/48/72 h

Score:

< 2.3

Irritant / corrosive response data:

A slight to well defined erythema and a very slight to slight oedema was seen in all rabbits 24 hours after application of the compound. A very slight oedema was still present in 3/6 rabbits at 72 hours. Also at this time 3/6 (No. 11, 14, 15) rabbits were showing slight desquamation of the epidermis at the application sites.
There was no significant difference between intact and abraded sites.
The primary irritation score was 1.6.

Animal N.	Reaction	Itact skin		Itact skin
		24 hrs	72 hrs	24 hrs	72 hrs
11 male	Erythema	1	0	1	0
13 male		1	0	1	0
15 male		1	0	1	0
12 female		1	0	1	0
14 female		2	0	2	0
16 female		1	0	1	0
11 male	Oedema	2	0	1	0
13 male		2	0	2	1
15 male		2	1	1	1
12 female		2	0	1	0
14 female		2	0	2	0
16 female		1	1	2	1

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Mild irritating

Executive summary:

The skin irritation potential of test item was assessed in six New Zealand White rabbits. The test was carried out according to the method given in the "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11 (February 1965).

A slight to well defined erythema and a very slight to slight oedema was seen in all rabbits 24 hours after application of the compound. A very slight oedema was still present in 3/6 rabbits at 72 hours. Also at this time 3/6 (No. 11, 14, 15) rabbits were showing slight desquamation of the epidermis at the application sites.

There was no significant difference between intact and abraded sites.

The primary irritation score was 1.6.

Conclusion

The scoring system criteria used to record the hardness of the skin reactions followed in the current test are the same as those reported into the OECD guideline, therefore it is possible to re-evaluate the results in accordance with the CLP Regulation (EC) No 1272/2008. Althought the observation time points were 24 and 72 hours, the mean scores for grading at 24, 48 and 72 hours can be reasonably estimated.

The mean values from gradings at 24, 48 and 72 hours were lower than lower than 2.3 for both erythema and oedema, in all the tested animals, both with skin shaved and scarified. Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC) No 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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The read across approach is detailed into the document attached into the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Code of Federal Regulation, title 16, Section 1500.42

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: W. Ostrop, Meerbusch.
- Weight at study initiation: 3.2 kg
- Housing: single cages.
- Diet: "mümmel z" (ssniff / Soest), ad libitum.
- Water: ad libitum.
- Acclimation period: 7 - 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 16 - 18 °C
- Humidity: 40 %
- Photoperiod: 12 hrs dark / 12 hrs light

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 µl of amorphous substance

Observation period (in vivo):

8 days

Number of animals or in vitro replicates:

6 rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: the eye was not rinsed after the application.

TOOL USED TO ASSESS SCORE: the assessment was performed with the help of an ophthalmoscope. The corneal epithelium was detected with the fluorescein test: installation of 1 drop of fluorescein solution in ophthalmological preparation directly on the cornea. After rinsing with a physiological solution of hydrochloric acid, horny yellow-green appears. UV light or cobalt blue filter.

SCORING SYSTEM
The eyes were examined and the grade of ocular reaction was recorded at 24, 48, 72 hours and 8 days.
The ocular reactions were scored by the method described by "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" FDA, 1959.

Mean Draize score
0 - 10 non-irritant
11 - 25 slightly irritant
26 - 56 moderately irritant
57 - 110 severe irritant

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, hemorrhage, 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: 6/6

Time point:

24/48/72 h

Score:

< 1

Irritation parameter:

iris score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 1

Irritation parameter:

conjunctivae score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 48 hrs

Irritation parameter:

chemosis score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 48 hrs

Irritant / corrosive response data:

Primary irritation index: 0.8, thus the substance can be considered as non-irritating.

Reaction	Animal N.	24 hrs	48 hrs	72 hrs	Mean 24/48/72 hrs
Opacity	1	0	0	0	0.00
Opacity	2	0	0	0	0.00
Opacity	3	0	0	0	0.00
Opacity	4	0	0	0	0.00
Opacity	5	0	0	0	0.00
Opacity	6	0	0	0	0.00
Iris	1	0	0	0	0.00
Iris	2	0	0	0	0.00
Iris	3	0	0	0	0.00
Iris	4	0	0	0	0.00
Iris	5	0	0	0	0.00
Iris	6	0	0	0	0.00
Conjunctival Redness	1	1	0	0	0.33
Conjunctival Redness	2	1	0	0	0.33
Conjunctival Redness	3	1	0	0	0.33
Conjunctival Redness	4	1	0	0	0.33
Conjunctival Redness	5	1	0	0	0.33
Conjunctival Redness	6	1	0	0	0.33
Conjunctival Chemosis	1	0	0	0	0.00
Conjunctival Chemosis	2	0	0	0	0.00
Conjunctival Chemosis	3	0	0	0	0.00
Conjunctival Chemosis	4	1	0	0	0.33
Conjunctival Chemosis	5	0	0	0	0.00
Conjunctival Chemosis	6	0	0	0	0.00

Individual reaction scores

Time	24 hrs						48hrs						72 hrs						8 days
Animal N.	1	2	3	4	5	6	1	2	3	4	5	6	1	2	3	4	5	6	1	2	3	4	5	6
Cornea
A. Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
B. Area	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
a = AxBx5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Iris
Iris (A)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
b = Ax5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Conjuntival
A. Redness	1	1	1	1	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
B. Chemosis	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
C. Discharge	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
c = (A+B+C)x2	2	2	2	4	2	2	0	0	0	0	0	0	0	0	0	0	0	0
a+b+c	2	2	2	4	2	2	0	0	0	0	0	0	0	0	0	0	0	0

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Non-irritating

Executive summary:

The procedures employed were that prescribed by Code of Federal Regulation, title 16, Section 1500.42. 100 µl of the test article were instilled into one eye of each animal, the other eye remaining untreated, served as a control.

The eyes were examined and the grade of ocular reaction was recorded at 24, 48, 72 hours and 8 days. The ocular reactions were scored by the method described by "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" FDA, 1959.

The orimary irritation index resulted to be 0.8, thus the substance can be considered as non-irritating.

Conclusion

Although the criteria for the evaluation of the eye irritation potential followed in the test are different from those reported into the OECD guideline, the scoring system is hte same. Because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC) No 1272/2008.

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 tested animals, thus the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

SKIN IRRITATION

There is no data about the skin irritation potential of Acid Orange 127; therefore, the available data on structural analogous Similar Substance 02 has been taken into consideration. The read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

The skin irritation potential of the Similar Substance 02 was assessed in six New Zealand White rabbits. The test was carried out according to the method given in the "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11 (February 1965). A slight to well defined erythema and a very slight to slight oedema was seen in all rabbits 24 hours after application of the compound. A very slight oedema was still present in 3/6 rabbits at 72 hours. Also at this time 3/6 (No. 11, 14, 15) rabbits were showing slight desquamation of the epidermis at the application sites. There was no significant difference between intact and abraded sites.

Althought the observation time points were 24 and 72 hours, the mean scores for grading at 24, 48 and 72 hours can be reasonably estimated. The mean values from gradings at 24, 48 and 72 hours were lower than lower than 2.3 for both erythema and oedema, in all the tested animals, both with skin shaved and scarified. Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC) No 1272/2008.

EYE IRRITATION

There is no data about the eye irritation potential of Acid Orange 127; therefore, the available data on structural analogous Similar Substance 01 has been taken into consideration. The read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

The procedures employed to evaluate the eye irritaiton potential of Similar Substance 01 were that prescribed by Code of Federal Regulation, title 16, Section 1500.42. 100 µl of the test article were instilled into one eye of each animal. The eyes were examined and the grade of ocular reaction was recorded at 24, 48, 72 hours and 8 days. The ocular reactions were scored by the method described by "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" FDA, 1959.

The orimary irritation index resulted to be 0.8, thus the substance can be considered as non-irritating.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.2 Skin corrosion/irritation section, skin Irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

The substance has been evaluated to be not skin irritating.

According to the CLP Regulation (EC) No 1272/2008 substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes) if, 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 has been evaluated to be not eye irritating.

In conclusion, the substance is not classified for skin/eye irritation, according to the CLP Regulation (EC) No 1272/2008.