Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Endpoint summary

Description of key information

Two primary skin irritation studies were performed on the test substance in rabbit and the substance was not considered to be an irritant in the rabbit. Two eye irritation tests were also performed in rabbits and the substance was considered to be irritating to eye. No data are available for respiratory irritation.

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:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

(no untreated skin areas to serve as the control; amount applied on skin not provided, exposure period is of 24 h; smaller area of application (i.e., approx. 2.5 cm² gauze pad))

Principles of method if other than guideline:

not applicable

GLP compliance:

not specified

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 1.5 to 2 kg
- Housing: The animals were housed individually in metal cages
- Diet: NAFAG, Gossau SG, rabbit food (ad libitum)
- Water: ad libitum
- Acclimation period: 4 d

ENVIRONMENTAL CONDITIONS
- Temperature: 20±1 °C
- Humidity: 55±5 %
- Photoperiod: 10 h light cycle day

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): No data
- Concentration (if solution): No data

Duration of treatment / exposure:

24 h

Observation period:

24, 48, 72 h, 4 and 7 d

Number of animals:

6 (3 males and 3 females)

Details on study design:

PROCEDURE:
- The test was carried out according to the method given in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO) which is similar to the OECD Guideline 404.
- Before treatment the entire back and the flank of the rabbits were shaved with an electric clipper and immediately before treatment the shaven skin on one side was slightly scarified.
- Gauze patches of 2.5 x 2.5 cm laden with the test substance were applied to the prepared abraded and intact skin.
- The patches were covered with an impermeable material and were fastened to the body of the rabbit with adhesive tape. The dressings were removed after a 24 h exposure. The skin reaction was appraised upon removal during an observation period of 7 d.

SCORING SYSTEM:
Dermal irritation was graded in each animal according to the following scoring scale:
- Erythema and eschar formation:
0 no erythema
1 very slight erythema (barely perceptible)
2 well-defined erythema
3 moderate to severe erythema
4 severe erythema (beet redness) to slight eschar formation (injuries in depth).
 
- Edema formation:
0 no edema
1 very slight edema (barely perceptible)
2 slight edema (edges of area well-defined by definite raising)
3 moderate edema (raised approximately 1 millimeter)
4 severe edema (raised more than 1 millimeter and extending beyond area of exposure).

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #1

Time point:

other: 24, 48, 72 h (mean)

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #2

Time point:

other: 24, 48, 72 h (mean)

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #3

Time point:

other: 24, 48, 72 h (mean)

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #4

Time point:

other: 24, 48, 72 h (mean)

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #5

Time point:

other: 24, 48, 72 h (mean)

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Remarks:

(intact skin)

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Remarks:

(intact skin)

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

Test substance was found to cause a slight irritation when applied to intact and abraded rabbit skin.
The calculated primary irritation index was 1.7.

Other effects:

none

Calculation of the primary skin irritation index:

Mean reaction score of 6 rabbits
Time after exposure (h)	erythema		edema
	intact skin	abraded skin	intact skin	abraded skin
24	0.3	2.3	0.3	2.3
72	0	0.83	0	0.83
Total	0.3	3.13	0.3	3.13	6.86

Primary irritation index = 6.86 : 4 = 1.7

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is considered to be not irritating to the skin.

Executive summary:

An in vivo study was conducted to evaluate the skin irritation potential of the test substance (at ca. 82 % purity) in Himalayan rabbits according to the method given in the Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO), which is similar to OECD Guideline 404 with few deviations. The test substance was placed on a 2.5 cm² gauze pad which was then applied to abraded as well as intact skin of rabbits for 24 h under occlusive conditions. After the application period, the dressing was removed and the intact and abraded sites were assessed for oedema and erythema. A further assessment was made at 48, 72 h, 4 and 7 d. Very slight erythema as well as edema were observed in two rabbits at 24 h. These symptoms were fully reversible within 48 h. The primary irritation index was calculated to be 1.7. Under the study conditions, the test substance was considered to cause slight irritation which was fully reversible within 48 h when applied to intact and abraded rabbit skin. However, it does not meet the threshold criteria for classification for skin irritation as required by the Regulation (EC) No. 1272/2008 (CLP), hence considered to be not irritating to the skin.

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:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

(observation period was up to 7 d instead of 21 d; eyes were not examined at 1 h after test substance application; clinical observations for systemic effects was not done)

Principles of method if other than guideline:

not applicable

GLP compliance:

not specified

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 1.5 to 2 kg
- Housing: The rabbits were caged singly in metal cages
- Diet: Standard rabbit food - NAFAG, Gossau SG (ad libitum)
- Water: ad libitum
- Acclimation period: 4 d

ENVIRONMENTAL CONDITIONS
- Temperature: 20±1 °C.
- Humidity: 55±5 %
- Photoperiod: 10 h (light cycle day)

Vehicle:

unchanged (no vehicle)

Controls:

other: the second eye on the treated animals served as control

Amount / concentration applied:

- Amount applied: 100 mg
- Concentration: Undiluted

Duration of treatment / exposure:

Unwashed eye: Duration of treatment same as observation period
Washed eye: 30 seconds

Observation period (in vivo):

The eyes were examined for any ocular reaction approximately 1, 2, 3, 4 and 7 d (i.e., 24, 48, 72, 96 and 168 h) after the treatment

Number of animals or in vitro replicates:

6 (i.e., 3 males and 3 females)

Details on study design:

PROCEDURE:
-The test was carried out in accordance with the procedure set out in the Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO) which is similar to OECD Guideline 405.
-Only rabbits with normal ophthalmic findings were used for the test.
-The test substance in an amount of 100 mg was inserted into the conjunctival sac of the left eye of the rabbits and the eyelids were gently closed for one second. The right eye was not treated and served as an untreated control.
-In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 mL of lukewarm water.
-The eye irritation was evaluated with a slit-lamp and was scored for each rabbit on 1, 2, 3, 4 and 7 d.

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Reversibility:

not specified

Remarks on result:

not measured/tested

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

13.9

Max. score:

80

Reversibility:

fully reversible

Remarks on result:

other: Individual scores were not cited.

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

10

Reversibility:

fully reversible

Remarks on result:

other: Individual scores were not cited.

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

14.4

Max. score:

20

Reversibility:

fully reversible

Remarks on result:

other: Individual scores were not cited.

Irritant / corrosive response data:

The irritation index was calculated to be 13.9 for the cornea, 0 for the iris and 14.4 for the conjunctivae. The test substance caused moderate overall eye irritation to rabbits. Rinsing the eyes following instillation increased the effect of the test substance.

Other effects:

none

Results of the eye irritation:

Unwashed eyes after application of test substance:

Days after Appl.	Rabbit 1 (M)			Rabbit 2 (M)			Rabbit 3 (M)
	Cornea	Iris	Conjunctiva	Cornea	Iris	Conjunctiva	Cornea	Iris	Conjunctiva
1	15	0	18	10	0	18	10	0	16
2	15	0	16	10	0	18	10	0	14
3	15	0	16	10	0	18	10	0	14
4	15	0	16	10	0	18	10	0	10
7	10	0	10	10	0	14	0	0	6

Cornea: 10.6; Iris: 0; Conjunctivae: 14.8

 

Eyes washed after application of test substance:

Days after Appl.	Rabbit 4 (F)			Rabbit 5 (F)			Rabbit 6 (F)
	Cornea	Iris	Conjunctiva	Cornea	Iris	Conjunctiva	Cornea	Iris	Conjunctiva
1	10	0	18	20	0	18	15	0	20
2	10	0	16	40	0	18	15	0	20
3	10	0	14	40	0	16	15	0	18
4	5	0	10	40	0	14	15	0	18
7	0	0	0	20	0	14	5	0	6

 

Cornea: 17.3; Iris: 0; Conjunctivae: 14

 

R (effect of rinsing) = unrinsed eyes/rinsed eyes (i.e., 8.4/10.4) = 0.8

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of the study, the test substance was considered to be moderately irritating to the eye.

Executive summary:

An in vivo study was conducted to evaluate the acute eye irritation potential of the test substance (at ca. 82 % purity) in rabbits according to the procedure described in the Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO), which is similar to OECD Guideline 405 but with few deviations. 100 mg of the test substance was placed into the conjunctival sac of the left eye of six rabbits (i.e., 3 male and 3 females). The right eye remained untreated and was used as control. In three of the six rabbits, washing was done with 10 mL of lukewarm water after 30 seconds of test substance instillation. Assessment of ocular damage/irritation was made with a slit-lamp and scored for each individual rabbit approximately on 1, 2, 3, 4 and 7 d following treatment. The total maximum score was 80 for the cornea, 10 for the iris, 20 for the conjunctivae and 110 for the entire eye. The irritation index was calculated to be 13.9, 0 and 14.4 for the cornea, iris and conjunctivae, respectively. Rinsing the eyes following instillation increased the effect of the test substance. Under the conditions of the study, the test substance was considered to be moderately irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two studies each are available for both skin and eye irritation.

 

Skin:

Both in vivo skin studies were in Himalayan rabbits according to the method given in the Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959) of the US Association of Food and Drug Officials (AFDO), which is similar to OECD Guideline 404 with few deviations. The test substance was placed on a 2.5 cm2 gauze pad which was then applied to abraded as well as intact skin of rabbits for 24 h under occlusive conditions. After the application period, the dressing was removed and the intact and abraded sites were assessed for oedema and erythema. A further assessment was made at 48, 72 h, 4 and 7 d. In the first study, Very slight erythema as well as edema were observed in two rabbits at 24 h. These symptoms were fully reversible within 48 h. The primary irritation index was calculated to be 1.7. Under the study conditions, the test substance was considered to cause slight irritation which was fully reversible within 48 h when applied to intact and abraded rabbit skin. In the second study, no skin reaction was observed in any of the treated animal. The primary irritation index was calculated to be 0. Under the study conditions, the test substance was considered to be non-irritating to rabbit skin.

 

Eye:

Both in vivo eye studies were completed in rabbits according to the procedure described in the Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO), which is similar to OECD Guideline 405 but with few deviations. 100 mg of the test substance was placed into the conjunctival sac of the left eye of six rabbits (i.e., 3 male and 3 females). The right eye remained untreated and was used as control. In three of the six rabbits, washing was done with 10 mL of lukewarm water after 30 seconds of test substance instillation. Assessment of ocular damage/irritation was made with a slit-lamp and scored for each individual rabbit approximately on 1, 2, 3, 4 and 7 d following treatment. The total maximum score was 80 for the cornea, 10 for the iris, 20 for the conjunctivae and 110 for the entire eye. In the first study the irritation index was calculated to be 13.9, 0 and 14.4 for the cornea, iris and conjunctivae, respectively. Rinsing the eyes following instillation increased the effect of the test substance. Under the conditions of the study, the test substance was considered to be moderately irritating to the eye. In the second study, the irritation index was calculated to be 8, 0 and 4.9 for the cornea, iris and conjunctivae, respectively. Under the conditions of the study, the test substance was considered to be slightly irritating to the eye.

Justification for selection of skin irritation / corrosion endpoint:
Most recent study available with the highest purity.

Justification for selection of eye irritation endpoint:
Most recent study with the highest purity.

Effects on eye irritation: moderately irritating

Justification for classification or non-classification

Based on the above stated assessment of the skin irritation potential of Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium the substance does not have to be classified as Irritating to skin according to Council Directive 2001/59/EC according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council. Based on the above assessment of the eye irritation potential of the substance does have to be classified as H319 Irritating to eyes according to Council Directive 2001/59/EC and Category 2 ( H319: Causes serious eye irritation) according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the council.