Chromate(3-), [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)][3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)]-, sodium

Endpoint summary

Description of key information

Not skin irritating.

Not eye 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:

From February 11 to 14, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG CH 4414 Fuellinsdorf / Switzerland.
- Age at study initiation: 15 - 16 weeks
- Weight at study initiation: 2.1 - 2.8 kg
- Housing: individually in stainless steel cages equipped with an automatic cleaning, and drinking system.
- Diet: ad libitum; pelleted standard Kliba 341, Batch 11/85 rabbit maintenance diet. Food was analysed for contaminants.
- Water: ad libitum; community tap water from Itingen. Water was analysed for contaminants.
- Acclimation period: four days under laboratory conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C.
- Humidity: 40 - 70 %.
- Air changes: air-conditioned with 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark, music/light period.

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The test article (0.5 g) was applied moistened with tap- water.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

2 male and 1 females

Details on study design:

TEST SITE
- Area of exposure: approximately 24 hours before treatment, the dorsal fur was shaved with an electric clipper, exposing an area of approximately 100 square centimetres (10 cm x 10 cm).
- Type of wrap if used: on test day 1, the test article was applied to the intact skin of the shaved, area. It was covered with a 3.0 cm x 3.0 cm patch of surgical gauze. The gauze was covered with aluminium foil and then covered with an occlusive dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: flushed with luk e warm tap water.
- Time after start of exposure: 4 hours after the application.

SCORING SYSTEM
The skin reaction was assessed according, to the QECD Guidelines for Testing of Chemicals, Section 4, number 404, pp. 3-4. The skin reaction was assessed at 1, 24, 48 and 72 hours intervals after the removal of the dressing, gauze patch and test article.

ERYTHEMA AND ESCHAR FORMATION
No erythema: 0
Very slight, erythema (barely perceptible): 1
Well defined erythemas_ 2
Moderate to severe erythema: 3
Severe erythema, (beet redness) to slight eschar formation (injuries irr depth): 4
Maximum possible score: 4

EDEMA FORMATION
No edema 0
Very slight, edema (barely perceptible): 1
Slight edema, (edges of area well defined by definite, raising): 2
Moderate edema (raised approximately 1 mm): 3
Severe edema (raised more than 1 mm and extending beyond area of exposure): 4
Maximum possible score: 4

Irritation parameter:

erythema score

Basis:

animal: #55, #56, #57

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #55, #56, #57

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

The test item showed a primary irritation score of 0 when applied to intact rabbit skin.
In the area, of application a black discoloration of the skin was observed which could be related to effects of the test article.
No destructions or irreversible alterations of the treated skin were observed. No corrosive effect was evident on the skin.

Other effects:

TOXIC SYMPTOMS / MORTALITY
No acute toxic symptoms mere observed in the animals during the test period, and no mortality occurred.

BODY WEIGHTS
The body weight gain of all rabbits was similar.

NECROPSY
Due to the results obtained, no macroscopic organ examination was indicated.

Animal/sex	Reaction	1 hr	24 hrs	48 hrs	72 hrs	Mean 24, 48 and 72 hrs
55 M	Erythema	0	0	0	0	0.00
56 M	Erythema	0	0	0	0	0.00
57 F	Erythema	0	0	0	0	0.00
55 M	Oedema	0	0	0	0	0.00
56 M	Oedema	0	0	0	0	0.00
57 F	Oedema	0	0	0	0	0.00

Interpretation of results:

other: Not classified as skin irritatnt according to the CLP Regulation (EC) No. 1272/2008.

Conclusions:

not irritating

Executive summary:

Method

The skin irritation/corrosion potential of the test item was assessed according to the OECD guideline 404 and EU Method B.4.

Results

Under the test conditions, the substance was found to cause a primary irritation score of 0 when applied to intact rabbit skin.

In the area of application, a black discoloration of the skin was observed which could be related to effects of the test article.

No corrosive effect had occurred on the skin at each measuring interval.

Conclusion

The mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in at least 2 of 3 animals for both erythema/eschar and oedema reactions.

In conclusion, the test item can be classified as non irritating, according to the CLP Regulation (EC 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 supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From February 07 to 17, 2005.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Justification for Read Across is detailed in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Amount / concentration applied:

A single application of 0.1 ml (51.3 mg) of the test material was administered to the non irrigated eye of three rabbits.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 rabbits

Details on study design:

Ocular reactions were recorded 1, 24, 48 and 72 hours after administration.

Irritation parameter:

cornea opacity score

Basis:

animal: #97, #104, #105

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #97, #104, #105

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal: #97, #104, #105

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal: #97, #104, #105

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

The test material produced iridial inflammation and minimal to moderate conjunctival irritation. All treated eyes appeared normal at the 72-hour observation.
Maximum Group Mean Score: 12.7. Mild irritant (Class 4 on A 1-8 scale)

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	97 Male					104 Male					105 Male
	IPR = 2					IPR = 3					IPR = 0+
Time After Treatment	1 hour	24 hours	48 hours	72 hours	Mean 24, 48 and 72 hrs	1 hour	24 hours	48 hours	72 hours	Mean 24, 48 and 72 hrs	1 hour	24 hours	48 hours	72 hours	Mean 24, 48 and 72 hrs
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	/	0	0	0	0	/	0	0	0	0	/
Score (E x F) x 5	0	0	0	0		0	0	0	0		0	0	0	0
IRITIS
D	0	0	0	0	0	1	0	0	0	0	1	0	0	0	0
Score (D x 5)	0	0	0	0	/	5	0	0	0	/	5	0	0	0	/
CONJUNCTIVAE
A = Redness	1 Sf	1 Sf	1 Sf	0 Sf	0.7	2 Sf	1 Sf	1 Sf	0 Sf	0.7	1 Sf	1 Sf	1 Sf	0 Sf	0.7
B = Chemosis	1	0	0	0	0	2	1	1	0	0.7	2	1	0	0	0.3
C = Discharge	1 Re	1Re	0	0	/	2 Re	1 Re	1 Re	0	/	2	1	1	0	/
Score (A + B + C) x 2	6	4	2	0		12	6	6	0		10	6	4	0
Total Score	6	4	2	0		17	6	6	0		15	6	4	0

IPR = Initial pain reaction

+ = One drop of local anaesthetic instilled into both eyes 1 to 2 minutes before treatment

Sf = Black staining of the fur around the treated eye

Re = Black residual test material

Interpretation of results:

other: Not classified as eye irritant according to the CLP Regulation (EC) No. 1272/2008.

Conclusions:

Not irritating.

Executive summary:

Method

The study was performed to assess the irritation of the test material to the eye of the New Zealand White rabbit (SPL Standard Test Method 560.09). The method followed OECD Guidelines for Testing of Chemicals (24 April 2002) No. 405 "Acute Eye Irritation/Corrosion" and Method B5 of Commission Directive 20041731EC (which constitutes Annex V of Council Directive 67/548IEEC).

A single application of 0.1 ml (51.3 mg) of the test material was administered to the non irrigated eye of three rabbits. Ocular reactions were recorded 1, 24, 48 and 72 hours after administration.

Results

The test material produced iridial inflammation and minimal to moderate conjunctival irritation. All treated eyes appeared normal at the 72-hour observation.

Maximum Group Mean Score: 12.7

Mild irritant (Class 4 on A 1-8 scale).

Conclusion

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 animals.

Therefore the substance does not meet the criteria to be classified as eye irritating.

In conclusion, the test item can be not classified as irritating, according to the CLP Regulation (EC 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

The skin irritation/corrosion potential of the Acid Brown 355b (ABr355b - EC: 280-689-4) was assessed according to the OECD guideline 404. In the area of application, a black discoloration of the skin was observed which could be related to effects of the test article. No corrosive effect had occurred on the skin at each measuring interval. The mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in at least 2 of 3 animals for both erythema/eschar and oedema reactions (Huntsman Textile Effects (Germany) GmbH, 1986).

The test was performed on the ABr355b (EC 280-689-4), trisodium salt: chromate(3-),[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)][3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)]-, trisodium.

The reference substance under registration is the Acid Brown 355 (ABr355 - EC 284-915-2), the form which can have a variable number of the sodium atoms. Thus, although it has been appointed a different EC number to the ABr355b trisodium salt form (EC 280-689-4), it can be considered as “included” into the structure variability of the ABr355 (EC 284-915-2). In this context, the use of the experimental data available on the ABr355b (EC 280-689-4), in order to assess the ABr355 (EC 284-915-2), can be considered as not a read across, but an evaluation on one of the possible forms of the ABr355.

Furthermore, based on the experience in the manufacture of the substance, the most frequent form is the trisodium salt.

EYE IRRITATION

Two disregarded studies on the ABr355 are available.

In one case the eye irritation potential was assessed according to the OECD guideline 405. Under the conditions, the test item was found to cause a slight, black discoloration of the cornea and conjunctivae of the treated eyes. No corrosion was observed at any of the measuring intervals.

The mean values from gradings at 24, 48 and 72 hours were equal to 1 for corneal opacity in two rabbits, but lower than 1 for iritis in all the rabbits tested and lower than 2 for both conjunctival redness and oedema, in 2 of 3 tested animals.

The positive response recorded in two out of three rabbits tested for corneal opacity at score 1, would lead to a classification in the category 2 for the Eye Irritation potential, according to the CLP Regulation (EC 1272/2008). Nevertheless the procedures described in the OECD guideline 405 report that "when testing solids, pastes, and particulate substances, the amount used should have a volume of 0.1 ml or a weight of not more than 100 mg. [...] If the solid test substance has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of 1 hour after treatment, the eye may be rinsed with saline or distilled water".

In this test the substance was not removed after 1 hour. Considering that the classification is strictly borderline this aspect may be discriminating.

It has been concluded that the results obtained in this test have to be confirmed in order to evaluate a correct classification.

The test was performed on the ABr355b (EC 280-689-4), trisodium salt: chromate(3-),[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)][3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)]-, trisodium.

The reference substance under registration is the ABr355 (EC 284-915-2), the form which can have a variable number of the sodium atoms. Thus, although it has been appointed a different EC number to the ABr355b trisodium salt form (EC 280-689-4), it can be considered as “included” into the structure variability of the ABr355 (EC 284-915-2). In this context, the use of the experimental data available on the ABr355b (EC 280-689-4), in order to assess the ABr355 (EC 284-915-2), can be considered as not a read across, but an evaluation on one of the possible forms of the ABr355.

Furthermore, based on the experience in the manufacture of the ABr355, the most frequent form is the trisodium salt.

The second study available on the ABr355 can not be used in order to evaluate the eye irritation potential of the ABr355 because there are no data available about the test procedures and about the scoring system criteria used to record the hardness of reactions recorded in the current test. Thus, the data are non assessable according to the CLP Regulation (EC 1272/2208).

Because the two studies available on the ABr355 are not useful to clarify the right eye irritation potential of the substance, a read across approach has been proposed. The Similar Substance 01 (Acid Brown 282 - ABr282 -) is a disodium salt and the main structural difference from the ABr355 is that it presents one sulphonated group less than the ABr355. It is expected that these differences have no significant impact on the eye irritation potential. Furthermore, there are no relevant differences between the typical composition of ABr355 and the composition of the ABr282 lot tested.

The read across approach has been further detailed in the report attached to the IUCLID section 13.

The study was performed to assess the irritation of the test material to the eye of the New Zealand White rabbit. The method followed OECD Guidelines for Testing of Chemicals (24 April 2002) No. 405. A single application of 0.1 ml (51.3 mg) of the test material was administered to the non irrigated eye of three rabbits. Ocular reactions were recorded 1, 24, 48 and 72 hours after administration. 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 animals. Therefore the substance does not meet the criteria to be classified as eye irritating.

Justification for selection of skin irritation / corrosion endpoint:

Study conducted according to internationally accepted testing guidelines and performed according to the GLP.

Justification for selection of eye irritation endpoint:

Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Justification for classification or non-classification

According to the CLP Regulation (EC 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.

In the key study, the mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in at least 2 of 3 animals for both erythema/eschar and oedema reactions.

According to the Regulation 1272/2008 (CLP) 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 mean values from gradings at 24, 48 and 72 hours recorded in the key study were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all animals in the key study.

In conclusion, the substance is not classified for the eye/skin irritation, according to the CLP Regulation (EC 1272/2008).