Benzenesulfonic acid, 4-chloro-2-[2-[4,5-dihydro-3-methyl-5-oxo-1-(3-sulfophenyl)-1H-pyrazol-4-yl]diazenyl]-5-methyl-, ammonium salt (1:2)

Endpoint summary

Description of key information

Two studies in rabbits were performed to evaluate irritating or corrosive properties of the test material to skin or eyes (EU guideline B.4 and B.5; GLP). Application of the test substance caused slight erythema of the skin, dulling of the cornea and mild conjuctival irritation. All reactions on skin and eyes had resolved within 2 days. Therefore, the substance is considered to be non-irritating.

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:

May - June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Froxfield (U.K.) Ltd., Petersfield, Hampshire, England
- Age at study initiation: approximately 13 to 14 weeks
- Weight at study initiation: 3.0 to 3.2 kg
- Housing: one animal per cage
- Diet: standard laboratory diet SDS Stanrab (P) Rabbit Diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: yes; duration not specified
- Microbiological status when known: not specified
- Method of randomisation in assigning animals to test and control groups: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 19°C
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 19
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To: not specified

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

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

VEHICLE
- Amount(s) applied (volume or weight with unit): A 0.5 g amount of the test substance was applied under a 25 mm x 25 mn gauze pad which had been moistened with 0.5 ml distilled water to one intact skin site on each animal.

Duration of treatment / exposure:

4h

Observation period:

4d

Number of animals:

3 females

Details on study design:

TEST SITE
- Area of exposure: dorso-lumbar region
- % coverage: not specified
- Type of wrap if used: gauze pad and "Elastoplast" elastic adhesive dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, the semi-occlusive dressing and gauze pad were removed and the treatment site was washed with warm water (30° to 40°C) to remove any residual test substance. The
treated area was blotted dry with absorbent paper.
- Time after start of exposure: 4h

OBSERVATION TIME POINTS
- Examination of the treated skin was made on Day 1 (i.e. approximately 60 minutes after removal of the dressings) and on Days 2, 3 and 4 (equivalent to 24, 48 and 72 hours after exposure).

SCORING SYSTEM:
Local dermal irritation was assessed using the prescribed numerical 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 (beet redness) to slight eschar formation (injuries in depth)
preventing erythema reading - 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 millimetre) - 3
Severe oedema (raised more than 1 millimetre and extending
beyond the area of exposure) - 4
Any other lesion not covered by this scoring system, was described.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

mean of all 3 animals

Time point:

other: approx. 60 minutes after removal of the dressing

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

erythema score

Basis:

mean

Remarks:

mean of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: approx. 60 minutes after removal of the dressing

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: approx. 60 minutes after removal of the dressing

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 60 minutes after removal of the dressing

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

edema score

Basis:

mean

Remarks:

mean of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Very slight erythema with or without very slight oedema was seen in all three animals on Day 1 only (score 1). All reactions has resolved by day 2.

Other effects:

There were no signs of toxicity or ill health in any rabbit during the observation period.

Tab. 1: Dermal reactions observed after application of the test substance

Rabbit number	E=Erythema 0=Oedema	Day
		1*	2	3	4
556	E 0	1 1	0 0	0 0	0 0
557	E 0	1 0	0 0	0 0	0 0
558	E 0	1 1	0 0	0 0	0 0

* Approximately 60 minutes after removal of the dressing

Interpretation of results:

GHS criteria not met

Conclusions:

A single semi-occlusive application of the test substance to intact rabbit skin for four hours elicited transient very slight dermal reactions which were fully reversible within 24 hours.

Executive summary:

A study was performed to assess the skin irritation potential of the test substance to the rabbit.

The method followed was that described in EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Mediod B.4. Acute toxicity (skin irritation). The study was performed under GLP conditions.

Three rabbits were each administered a single dermal dose of 0.5 g of the test substance and observed for four days.

A single semi-occlusive application of the test substance to intact rabbit skin for four hours elicited transient very slight dermal reactions. All reactions had resolved by Day 2.

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

Study period:

June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Froxfield (U.K.) Ltd., Petersfield, Hampshire, England or Charles River (UK) Ltd., Margate, Kent, England
- Age at study initiation: approximately 12 to 19 weeks
- Weight at study initiation: 2.7 to 3.9 kg
- Housing: one animal per cage
- Diet: standard laboratory diet SDS Stanrab (P) Rabbit Diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: yes; duration not specified
- Microbiological status when known: not specified
- Method of randomisation in assigning animals to test and control groups: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 19°C
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 19
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To: not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 76 mg (occupying a volume of 0.1 ml)

Duration of treatment / exposure:

single application

Observation period (in vivo):

7d

Number of animals or in vitro replicates:

3 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM:
Examination of the eyes was made after 1 hour and 1, 2, 3 (equivalent to 24, 48 and 72 hours after instillation), 4 and 7 days after instillation.

Ocular irritation was assessed using the prescribed numerical system:
- Cornea:
Opacity: degree of density (area most dense taken for reading)
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 areas, details of iris slightly obscured - 2
Nacreous areas, no details of iris visible, size of pupil barely discernible - 3
Opaque cornea, iris not discernible through the opacity - 4
- Iris:
Normal - 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, any 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

- Conjunctivae
Redness (refers to the most severe reading of palpebral and bulbar conjunctivae, as compared to the control eye)
Blood vessels normal - 0
Some blood vessels definitely hyperaemic (injected) - 1
Diffuse, crimson colour, individual vessels not easily discernible - 2
Diffuse beefy red - 3
Chemosis (lids and/or nictating membranes)
No swelling - 0
Any swelling above normal (includes nictating membranes) - 1
Obvious swelling with partial eversion of lids - 2
Swelling with lids about half-closed - 3
Swelling with lids more than half-closed - 4

Any other lesion not covered by this scoring system, was described.

TOOL USED TO ASSESS SCORE: handheld light

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

mean of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

mean of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

mean of all 3 animals

Time point:

24/48/72 h

Score:

0.333

Max. score:

3

Reversibility:

fully reversible within:

Remarks:

2 d

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

mean of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 1 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within:

Remarks:

24 h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: 1 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within:

Remarks:

24 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: 1 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within:

Remarks:

24 h

Irritant / corrosive response data:

Dulling of the normal lustre of the cornea was seen in two animals one hour after instillation only.
No other corneal damage or iridial inflammation was observed.
Considerable swelling with partial eversion of the eyelids was seen in one animal one hour after instillation only. Temporary mild conjunctival reactions were seen in the remaining animals.
The eyes were normal two days after instillation

Other effects:

There were no signs of toxicity or ill health in any rabbit during the observation period.

Tab. 1: Ocular reactions observed after instillation of the test substance

Rabbit number	Region of eye		One hour	Day after instillation
				I23 47
543	Cornea		D	000 00
	Iris		0	00 0 0 0
	Conjunctiva	Redness	I	I0 0 0 0
		Chemosis	I	00000
580	Cornea		D	0 0 0 0 0
	Iris		0	0 000 0
	Conjunctiva	Redness	I	I000 0
		Chemosis	2	0 0 0 0 0
582	Cornea		0	0 0 0 0 0
	Iris		0	0 0 0 0 0
	Conjunctiva	Redness	I	1000 0
		Chemosis	1	00 0 00

Interpretation of results:

GHS criteria not met

Conclusions:

Instillation of the test substance into the rabbit eye elicited dulling of the cornea and mild transient conjunctival irritation. All reaction has resolved within 2d. The substance is therefore considered to be non-irritating.

Executive summary:

A study was performed to assess the eye irritation potential of the test substance to the rabbit.

The method followed was that described in EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Mediod B.5. Acute toxicity (eye irritation). The study was conducted under GLP conditions.

Three rabbits were each administered a single ocular dose of 76 mg of the test substance and observed for seven days after instillation. A single instillation of the test material into the eye of the rabbit elicited dulling of the cornea and transient mild conjunctival irritation. All reactions had resolved 2 days after instillation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

A study was performed to assess the skin irritation potential of the test substance to the rabbit. The method followed was that described in EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Method B.4. Acute toxicity (skin irritation). The study was performed under GLP conditions.

Three rabbits were each administered a single dermal dose of 0.5 g of the test substance and observed for four days.

A single semi-occlusive application of the test substance to intact rabbit skin for four hours elicited transient very slight dermal reactions. All reactions had resolved by Day 2.

Eye irritation:

A study was performed to assess the eye irritation potential of the test substance to the rabbit. The method followed was that described in EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Method B.5. Acute toxicity (eye irritation). The study was conducted under GLP conditions.

Three rabbits were each administered a single ocular dose of 76 mg of the test substance and observed for seven days after instillation.

A single instillation of the test material into the eye of the rabbit elicited dulling of the cornea and transient mild conjunctival irritation. All reactions had resolved 2 days after instillation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. In animal studies with rabbits very slight reactions were observed which fully resolved within 2 days after application of the test substance. As a result the substance is not considered to be classified for irritation under Regulation (EC) No. 1272/2008.