Trisodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)][2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzenesulphonato(3-)]chromate(3-)

Endpoint summary

Description of key information

Not skin irritating

Eye Irrit. 2 (H319)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Spolkem breeder of Hrace Králové.
- Weight at study initiation: above 2.5 kg.
- Housing: individually housed, in commercially metal cages (supplied by s.p. Velaz Prague), without litter and climatic control.
- Diet: standard granular commercial food for rabbits, KO-16 provided by s.p. Velaz Prague.
- Water: drinking water, ad libitum.
Cleaning and disinfection of premises menagerie were made at dates determined, according to the standard operating procedures and compliance regime measures.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C.
- Humidity: 50 ± 15 %.
- Photoperiod: 12 hrs dark / 12 hrs light, by fluorescent lamp.

Type of coverage:

occlusive

Preparation of test site:

other: intact and abraded skin

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
- Amoun applied: 0.5 g

Duration of treatment / exposure:

24 hours

Observation period:

72-hours

Number of animals:

3 animals

Details on study design:

TEST SITE
- Area of exposure: 24 hours after the test material application, rabbits were shaved (skin area of ​​6 x 6 cm) on the left and right side of the lateral surface of the abdominal wall localized in the region epigastrium, mesogastrium and hypogastrium. Left side was intact, while the right side was scarificated.
- Type of wrap if used: test material was applied to skin on left side and covered with a gaude. Then water was applied to a gauze using a syringe, in order to obtain an aqueous paste. Furthermore on gauze were enclosed aluminium foil and cellulose wadding. The coverage was fixed by technical tape.

SCORING SYSTEM: OECD guideline 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 slight eschar formation (injuries in depth): 4
Maximum possible: 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 area of exposure): 4
Maximum possible: 4

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

< 2.3

Max. score:

4

Remarks on result:

other: both intact and abraded skin

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

< 2.3

Max. score:

4

Remarks on result:

other: both intact and abraded skin

Irritant / corrosive response data:

Primary dermal irritation index (PDII): 0.5
Degree of dermal irritation: 2. Weakly irritant.
INTACT SKIN
Rabbit 1: a well-defined erythema and oedema.
Rabbit 2: very slight erythema and oedema very weak.
Rabbit 3: very slight erythema and oedema well defined.
For 24 hours were found very slight erythema and oedema very weak. At 48 and 72 hours no functional macroscopical, nor patomorphological changes were recorded.

SCARIFIED SKIN
Rabbit 1: a well-defined erythema and oedema.
Rabbit 2: very slight erythema and oedema very weak.
Rabbit 3: very slight erythema and oedema well defined.
At 48 and 72 hours no functional macroscopical, nor patomorphological changes were recorded.

Reactions observed

N. animal cage	Reaction	Reaction after				Mean 24, 48 and 72 hrs
		4 hrs	24 hrs	48 hrs	72 hrs
Intact skin
1	Erythema	2	0	0	0	0.00
	Oedema	2	1	0	0	0.33
2	Erythema	1	0	0	0	0.00
	Oedema	1	1	0	0	0.33
3	Erythema	1	0	0	0	0.00
	Oedema	2	0	0	0	0.00
Abraded skin
1	Erythema	2	0	0	0	0.00
	Oedema	2	0	0	0	0.00
2	Erythema	1	1	0	0	0.33
	Oedema	1	1	0	0	0.33
3	Erythema	2	1	0	0	0.33
	Oedema	3	2	0	0	0.67

Interpretation of results:

not classified

Remarks:

Migrated information according to the CLP Regulation (EC 1272/2008) Criteria used for interpretation of results: EU

Conclusions:

Not irritating.

Executive summary:

The skin irritation potential of the substance was determined by the skin irritation test, according to the OECD guideline 404.

0.5 g of test material were applied to intact and scarified skin of rabbits and removed after 24 hours. Observations were made for 72 hours.

Primary dermal irritation index (PDII) was determined to be 0.5 and the degree of dermal irritation was stated at 2.

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 according to the CLP Regulation (EC 1272/2008), because the raw tables are included into the study report.

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions (in both intact and abraded skin).

Conclusion

The test item can be not classified as skin 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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Spolkem breeder of Hrace Králové.
- Weight at study initiation: above 2.5 kg.
- Housing: individually housed, in commercially metal cages (supplied by s.p. Velaz Prague), without litter and climatic control.
- Diet: standard granular commercial food for rabbits, KO-16 provided by s.p. Velaz Prague.
- Water: drinking water, ad libitum.
Cleaning and disinfection of premises menagerie were made at dates determined, according to the standard operating procedures and compliance regime measures.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C.
- Humidity: 50 ± 15 %.
- Photoperiod: 12 hrs dark / 12 hrs light, by fluorescent lamp.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 g

Observation period (in vivo):

72 hours (observations were made at 1, 24, 48 and 72 hours after installation of test material).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: water.
- Time after start of exposure: 24 hours. Areas with the loss of corneal tissue are intensely coloured and the progressive rinsing of the dye in the damaged areas require more time than the place intact.

TOOL USED TO ASSESS SCORE: 2 % sodium salt, fluorescein. Reactions after 48 hours were evaluated without fluorescein. Reactions after 72 hours were evaluated with 2 % aqueous fluorescein solution.

SCORING SYSTEM

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:
Normal: 0
Folds above normal, congestion, swelling, circum cornea 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, haemorrhage, 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 canthus 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:

mean

Time point:

24/48/72 h

Score:

> 1

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

>= 1

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

> 2

Max. score:

4

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

> 2

Max. score:

4

Irritant / corrosive response data:

Primary irritation score: 63.3
Degree of irritation: 4

The following effects were recorded at 1, 24, 48 and 72 hours: visual examination was found massive purulent discharge from the corner of eye, blepharospasm, chemosis coniunctivae, diffuse injection of the lids and bulbar conjunctiva, cornea in quadrant rich milky turbid-leucoma with a total loss of transparency, iris obscured with loss of reaction to light.

Reactions observed

N. animal cage	Assessment	Cornea		Iritis	Conjunctival
		Reaction	Area		Congestion	Swelling	Discharge
1	1	1	4	2	2	3	3
	24	2	4	1	2	3	3
	48	2	4	1	2	2	3
	72	2	4	2	2	2	3
	Mean 24, 48 and 72 hrs	2.00		1.33	2.00	2.33
2	1	1	4	1	2	3	3
	24	2	4	1	2	3	3
	48	2	4	1	2	2	3
	72	1	4	1	2	2	3
	Mean 24, 48 and 72 hrs	1.67		1.00	2.00	2.33
3	1	1	4	2	2	3	3
	24	2	4	1	2	3	3
	48	2	4	1	2	1	3
	72	2	4	1	1	2	2
	Mean 24, 48 and 72 hrs	2.00		1.00	1.67	2.00

Interpretation of results:

other: Eye Irrit. 2 (H319), according to the CLP Regulation (EC 1272/2008)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Eye Irrit. 2, H319.

Executive summary:

The skin irritation potential of the substance was determined by the skin irritation test, according to the OECD guideline 404.

0.5 g of test material were applied to intact and scarified skin of rabbits and removed after 24 hours. Observations were made for 72 hours.

The substance resulted strong irritant. Primary irritation score was stated at 63.3 and the degree of irritation as 4.

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 used for the cornea opacity and degree of density, for the iris and conjunctivae redness and chemosis is the same of that reported into the OECD, but the timing for the removal of the substance are very different (24 hours in the current study and 1 hour in the OECD). Nevertheless, because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC 1272/2008).

The mean values from gradings at 24, 48 and 72 hours was:

- higher than 1 in all the animals tested for corneal opacity. Nevertheless in none was equal/higher than 3.

- equal than 1 in two rabbits and 1.33 in the last one for iritis

- higher than 2 in two rabbit, for conjunctival redness.

- higher than 2 in two animals, for conjunctival chemosis

Information are not available about the reversibility of the effects recorded. A relevant aspect, that may be determinant in the assessment, is that the solid substance tested was removed after 24 hours after application, instead of 1 hour as required by the OECD guideline 405 updated version 2012. Thus, it can not be excluded that the irritation potential determined by the powder form may contribut to the reactions observed.

In conclusion, it is considered appropriate a classification as eye irritant, category 2 (H319), according to the CLP Regulation (EC 1272/2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

The skin irritation potential of the substance was determined by the skin irritation test, according to the OECD guideline 404. 0.5 g of test material were applied to intact and scarified skin of rabbits and removed after 24 hours. Observations were made for 72 hours.

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions (in both intact and abraded skin) (Synthesia, a.s. - SBU PaB, 1994).

EYE IRRITATION

The skin irritation potential of the substance was determined by the skin irritation test, according to the OECD guideline 404. 0.5 g of test material were applied to intact and scarified skin of rabbits and removed after 24 hours. Observations were made for 72 hours.

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 used for the cornea opacity and degree of density, for the iris and conjunctivae redness and chemosis is the same of that reported into the OECD, but the timing for the removal of the substance are very different (24 hours in the current study and 1 hour in the OECD). Nevertheless, because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC 1272/2008).

The mean values from gradings at 24, 48 and 72 hours was:

- higher than 1 in all the animals tested for corneal opacity. Nevertheless in none was equal/higher than 3.

- equal than 1 in two rabbits and 1.33 in the last one for iritis

- higher than 2 only in one rabbit, for conjunctival redness.

- higher than 2 in two animals, for conjunctival chemosis

Information are not available about the reversibility of the effects recorded. A relevant aspect, that may be determinant in the assessment, is that the solid substance tested was removed after 24 hours after application, instead of 1 hour as required by the OECD guideline 405 updated version 2012. Thus, it can not be excluded that the irritation potential determined by the powder form may contribut to the reactions observed (Synthesia, a.s. - SBU PaB, 1994).

Justification for selection of skin irritation / corrosion endpoint:

Study conducted according to internationally accepted testing guideline.

Justification for selection of eye irritation endpoint:

Study conducted according to internationally accepted testing guideline.

Effects on eye irritation: irritating

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.

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions (in both intact and abraded skin).

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 was:

- higher than 1 in all the animals tested for corneal opacity. Nevertheless in none was equal/higher than 3.

- equal than 1 in two rabbits and 1.33 in the last one for iritis

- higher than 2 only in one rabbit, for conjunctival redness.

- higher than 2 in two animals, for conjunctival chemosis

In conclusion, the substance is not classified for the skin irritation and it is classified as eye irritant, category 2 (H319), according to the CLP Regulation (EC 1272/2008).