Tetrasodium 4-[[3,5-bis[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]-2,4(or 2,6)-dihydroxyphenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000, March

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

other: Real Decree 363/1995 and Real Decree 1078/93

Principles of method if other than guideline:

Real Decree 363/1995 is the transposition of Directive 67/548/EEC in Spanish legislation

GLP compliance:

no

Remarks:

Centre Certificated ISO 9001 by National Accreditation Programme (certificate N. 98 - 1101)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: authorized provider
- Weight: from 2.012 to 2.113 g
- Housing: inoxidable steel individual cage, Techiplast. Rabbit were maintained in cages for the total duration of the test
- Diet: daily 150 g of special fodder for experimental rabbit, by authorized provider
- Water: ad libitum, treated and filtrated
- Marking: tatoo on the upper side of ear
- Acclimatation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 25
- Air changes: 15 prefiltered air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 regulated by timer

USED GENERAL MATERIALS:
Balance: COBOS D 6000-SX
Vinyl/latex gloves mono-used
Syringes: 1 mL of volume
Shaver: electric AESCULAP mod. Favorita II
Gauzes dressing: Melolin or equivalent authorized
Adhesive bandage
Bandage: hypoalergenic adhesive Fixomull Seretch or equivalent authorized

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

physiological saline

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
Amount applied: 0.5 g of solid substance humidified with water for assuring a good contact to the skin

Duration of treatment / exposure:

4 hours

Observation period:

Observation at: 1, 24, 48 and 72 hours

Number of animals:

3 males

Details on study design:

TEST SITE
- Area shaved: retro-scapular side in the area around the vertebral column (20 cm of length and 13 cm of height).
- Area of exposure: 6 cm^2
- Type of wrap if used: gauzed dressing closed by adhesive bandage, covered with adhesive hypoalergenic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: water or specific solvent

SCORING SYSTEM:
Erythema and Eschar Formation
No erythema: 0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema: 4
Maximum possible: 4

Edema 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 area of exposure): 4

If reactions are not reversible until 72h, it could be necessary an observation period of a maximum 14 days

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0.44

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

0

Reversibility:

fully reversible

Irritant / corrosive response data:

Observation: in rabbits 1 and 2, at 72 h lesions were fully reversible, while rabbit 3 does not show any irritating effects.

Erythema and edema

Time of observation		Rabbit 1		Rabbit 2		Rabbit 3
		Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
	1h	1	1	1	1	0	0
	24h	1	1	1	0	0	0
	48h	1	1	1	0	0	0
	72h	0	0	0	0	0	0

Media erythema: 0.44

Media edema: 0

Interpretation of results:

GHS criteria not met

Conclusions:

Not skin irritant

Executive summary:

The susbtance was tested for skin irritation according to the Real Decree 363/1995 is the transposition of Directive 67/548/EEC in Spanish legislation

All lesions observed were all completely reversible maximum in 72 hours and one animal does not show any irritating effects. The tested substance is not considered as skin irritant.

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:

experimental study

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

Qualifier:

according to guideline

Guideline:

other: Real Decree 363/1995 and Real Decree 1078/93

Principles of method if other than guideline:

Real Decree 363/1995 is the transposition of Directive 67/548/EEC in Spanish legislation

GLP compliance:

no

Remarks:

Centre Certificated ISO 9001 by National Accreditation Programme (certificate N. 98 - 1101)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: certificated provider
- Sex: males
- Housing: individual inoxidable steel cages, Tecniplast
- Diet: daily 150 g of special fodder for experimental rabbits, supplied by an authorized provider
- Water: automatic ad libitum, treated and filtered
- Marking: tatoo on the upper side of ear
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 25
- Air changes (per hr): 15 changes of pre-filtered air per hour
- Photoperiod (hrs dark / hrs light): 12/12

Other materials used:
Balance: COBOS D 6000-SX
Gloves: vinyl and latex gloves
Syringes: 1 mL of volume
Manual lamp
Wash bottle with saline solution
Colircusi fluorescein or equivalent authorized

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: in case of liquid substance were administrated 0.1 mL, in case of solid substance 0.1 g

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3 males

Details on study design:

TOOL USED TO ASSESS SCORE: light of a manual lamp for examing the reactions and fluorescein for evaluating corneal lesions

SCORING SYSTEM:
Cornea
Opacity: degree of density (readings should be taken from most dense area)*
No ulceration or opacity: 0 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre);
details of iris clearly visible: 1
Easily discernible translucent area; details of iris slightly obscured: 2
Nacrous area; no details of iris visible; size of pupil barely discernible: 3
Opaque cornea; iris not discernible through the opacity: 4
Maximum possible: 4
* The area of corneal opacity should be noted

Iris
Normal: 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia;
or injection; iris reactive to light (a sluggish reaction is considered to be an effect: 1
Hemorrhage, gross destruction, or no reaction to light: 2
Maximum possible: 2

Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)
Normal: 0
Some blood vessels hyperaemic (injected): 1
Diffuse, crimson colour; individual vessels not easily discernible: 2
Diffuse beefy red: 3
Maximum possible: 3

Chemosis
Swelling (refers to lids and/or nictating membranes)
Normal: 0
Some swelling above normal: 1
Obvious swelling, with partial eversion of lids: 2
Swelling, with lids about half closed: 3
Swelling, with lids more than half closed: 4
Maximum possible: 4

DEFINITIONS:
1. Eye irritation is the production of changes in the eye following the application of a test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.
2.Eye corrosion is 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.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 7 days for all rabbits

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0.67

Max. score:

2

Reversibility:

fully reversible

Remarks:

Rabbit 1: 72 hours; Rabbit 2: 48 hours; Rabbit 3: 7 days

Irritation parameter:

conjunctivae score

Remarks:

Congiuntival erythema

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 1.89

Max. score:

3

Reversibility:

fully reversible

Remarks:

within 14 days for rabbit 1 and 3; within 7 days for rabbit 2

Irritation parameter:

chemosis score

Remarks:

Congiuntival edema

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 1.44

Max. score:

4

Reversibility:

fully reversible

Remarks:

wihtin 7 days for all rabbits

Results

	Rabbit 1						Rabbit 2								Rabbit 3
	1h	24h	48h	72h		7d	1h		24h		48h		72h	7d	1h	24h	48h	72h	7d
Corneal opacity	0	1	1	1		0	0		1		1		1	0	0	1	1	1	0
Iris	0	1	1	0		0	0		1		0		0	0	0	1	1	1	0
Conjunctival erythema	0	2	2	2		1	0		2		2		1	0	0	2	2	2	1
Conjuntival oedema	3	2	2	1		0	3		2		1		1	0	3	2	1	1	0

Interpretation of results:

GHS criteria not met

Conclusions:

Some irritating effects on all rabbits fully reversible within 14 days.

Executive summary:

The substance was tested for eye irritation according to the Real Decree 363/1995 is the transposition of Directive 67/548/EEC in Spanish legislation. Based on test results, the susbtance needs to be considered as eye irritant and required the hazard statement H319, Causes serious eye irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

No effect on skin was observed in the skin irritaion test on rabbits. Some effects on eye of all the treated rabbits was observed, fully reversible within 14 days.

Justification for selection of skin irritation / corrosion endpoint:
study well reported

Effects on eye irritation: irritating

Justification for classification or non-classification

The CLP Regulation inidcates the following values for the classification fo skin and eye irritation.

Skin irritant cat 2:

1) Mean value of ≥ 2,3 - ≤ 4,0 for erythema/ eschar or for oedema in at least 2 of 3 tested animals from gradings 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

2) 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

3) 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.

Eye irritant Cat 2: at least in 2 of 3 tested animals, a positive response of:

1) corneal opacity ≥ 1

and/or

2) iritis ≥ 1

and/or

3) conjunctival redness ≥ 2

and/or

4) conjunctival oedema (chemosis) ≥ 2 5calculated 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.

Based on the test results the susbtance is considered to be:

- not skin irritant

- eye irritant

The classification based on Regulation 1272/2008 is Eye Irritant Cat. 2, H319, Causes serious eye irritation.