Potassium sodium 4,4'-bis[6-anilino-4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines, well documented and the results scientifically acceptable. Some minor details missing.

Principles of method if other than guideline:

The procedure adopted was the patch-test technique 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).

GLP compliance:

no

Remarks:

Pre GLP.

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: WIGA Versuchstierzuchtanstalt Sulzfeld, Germany.
- Age at study initiation: adult.
- Weight at study initiation: 1.5 to 2.
- Housing: individually housed in metal cages.
- Diet: ad libitum, standard rabbit food NAFAG, Gossau SG.
- Water: ad libitum.
- Acclimation period: for a minimum of 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: constant room temperature of 22 ± 1 °C
- Humidity: relative humidity of 55 ± 5 %
- Photoperiod: 10 hrs light cycle day.

Type of coverage:

occlusive

Preparation of test site:

other: abraded and intact shaved skin.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

Gauze of 2.5 x 2.5 cm laden with 0.5 ml of the test substance were applied to the prepared abraded and intact skin.

Duration of treatment / exposure:

The dressing were removed after a 24 hrs application.

Observation period:

7 days

Number of animals:

3 males and 3 females.

Details on study design:

TEST SITE
- Area of exposure: 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.
- Type of wrap if used: patches were covered with an impermeable material and were fastened to the body of the rabbit with adhesive tape.

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:

animal: 6/6

Time point:

24/48/72 h

Score:

< 2.3

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: intact and abraded skin

Irritation parameter:

edema score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 2.3

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: intact and abraded skin

Irritant / corrosive response data:

Primary irritation index was estimated to be 0.83; minimal irritating.

Evaluation of skin reaction

Animal	Skin site	Parameter	hrs			days		Mean 24, 48 and 72 hrs
			24	48	72	4	7
1M	Intact skin	Erythema	0	0	0	0	0	0.00
		Oedema	0	0	0	0	0	0.00
	Abraded skin	Erythema	1	1	0	0	0	0.67
		Oedema	1	1	0	0	0	0.67
2M	Intact skin	Erythema	0	0	0	0	0	0.00
		Oedema	0	0	0	0	0	0.00
	Abraded skin	Erythema	1	1	1	1	0	1.00
		Oedema	1	1	1	1	0	1.00
3M	Intact skin	Erythema	0	0	0	0	0	0.00
		Oedema	0	0	0	0	0	0.00
	Abraded skin	Erythema	1	1	1	0	0	1.00
		Oedema	1	1	1	0	0	1.00
4F	Intact skin	Erythema	0	0	0	0	0	0.00
		Oedema	0	0	0	0	0	0.00
	Abraded skin	Erythema	1	1	1	0	0	1.00
		Oedema	1	1	1	0	0	1.00
5F	Intact skin	Erythema	0	0	0	0	0	0.00
		Oedema	0	0	0	0	0	0.00
	Abraded skin	Erythema	1	1	1	1	0	1.00
		Oedema	1	1	1	1	0	1.00
6F	Intact skin	Erythema	0	0	0	0	0	0.00
		Oedema	0	0	0	0	0	0.00
	Abraded skin	Erythema	1	1	0	0	0	0.67
		Oedema	1	1	0	0	0	0.67

Interpretation of results:

not irritating

Remarks:

Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU

Conclusions:

Not irritating.

Executive summary:

Method

The skin irritation potential was assessed following the procedure of the patch-test technique 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).

Results

Primary irritation index was estimated to be 0.83, therefore the substance resulted minimal irritating.

Discussion and conclusion

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. Only the intact skin reactions were considered.

Mean values from grading at 24, 48 and 72 hours after patch removal were 0 in all animals for both erythema/eschar and oedema reactions.

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

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:

other: Study conducted according to internationally accepted testing guidelines, well documented and the results scientifically acceptable. Some minor details missing.

Principles of method if other than guideline:

The procedure adopted was the patch-test technique 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).

GLP compliance:

no

Remarks:

Pre GLP.

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: WIGA Versuchstierzuchtanstalt Sulzfeld, Germany.
- Age at study initiation: adult.
- Weight at study initiation: 1.5 to 2.
- Housing: individually housed in metal cages.
- Diet: ad libitum, standard rabbit food NAFAG, Gossau SG.
- Water: ad libitum.
- Acclimation period: for a minimum of 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: constant room temperature of 22 ± 1 °C
- Humidity: relative humidity of 55 ± 5 %
- Photoperiod: 10 hrs light cycle day.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 ml

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

Six animals

Details on study design:

APPLICATION
The test material in an amount of 0.1 ml was inserted into the conjunctival sac of the left eye of the 6 rabbits and the lids were gently closed for a few seconds. The right eye was not treated and served as an untreated control.

REMOVAL OF TEST SUBSTANCE
- Washing: in 3 of the 6 rabbits the treated eye was flushed with 10 ml of physiological saline.
- Time after start of exposure: approximately 30 seconds after treatment.

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

IRIS:
A Values
Normal: 0
Folds above normal, congestion, swelling, circumcornea 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, hemorrhage, gross destruction (any or all of these): 2
A x 5; Maximum possible score = 10

CONJUNCTIVAE:
A Redness (refers to palpebral 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

TOOL USED TO ASSESS SCORE: eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7.

Irritation parameter:

cornea opacity score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritation parameter:

iris score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritation parameter:

conjunctivae score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritation parameter:

chemosis score

Basis:

animal: 6/6

Time point:

24/48/72 h

Score:

< 2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritant / corrosive response data:

The primary irritation index in unrinsed eyes was estimated to be 0.
The primary irritation index in rinsed eyes was estimated to be 0.

Rabbit eye irritation scores

Animal	Eye	Parameter	hrs			days
			24	48	72	4	7
		Cornea
1	Rinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
	Unrinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
2	Rinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
	Unrinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
3	Rinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
	Unrinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
4	Rinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
	Unrinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
5	Rinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
	Unrinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
6	Rinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
	Unrinsed	Opacity	0	0	0	0	0
		Area involved	0	0	0	0	0
		Iris
1	Rinsed		0	0	0	0	0
	Unrinsed		0	0	0	0	0
2	Rinsed		0	0	0	0	0
	Unrinsed		0	0	0	0	0
3	Rinsed		0	0	0	0	0
	Unrinsed		0	0	0	0	0
4	Rinsed		0	0	0	0	0
	Unrinsed		0	0	0	0	0
5	Rinsed		0	0	0	0	0
	Unrinsed		0	0	0	0	0
6	Rinsed		0	0	0	0	0
	Unrinsed		0	0	0	0	0
		Conjunctiva
1	Rinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
	Unrinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
2	Rinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
	Unrinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
3	Rinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
	Unrinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
4	Rinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
	Unrinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
5	Rinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
	Unrinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
6	Rinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0
	Unrinsed	Redness	0	0	0	0	0
		Chemosis	0	0	0	0	0
		Discharge	0	0	0	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU

Conclusions:

Not irritating

Executive summary:

Method

The eye irritation potential was assessed following the procedure of the patch-test technique 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).

Results

The primary irritation index in unrinsed eyes was estimated to be 0.

The primary irritation index in rinsed eyes was estimated to be 0.

Therefore, the substance resulted not irritating.

Discussion and conclusion

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 corea 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 (30 seconds in the current study and 1 hours 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 for both rinsed and unrinsed eyes were 0 in all animals for all the reactions scored.

In conclusion, the test item can be classified as NON irritating, according to the CLP (EC 1272/2008) Regulation.

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 potential was assessed following the procedure of the patch-test technique 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). Primary irritation index was estimated to be 0.83, therefore the substance resulted minimal irritating. 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. Only the intact skin reactions were considered.

Mean values from grading at 24, 48 and 72 hours after patch removal were 0 in all animals for both erythema/eschar and oedema reactions.

In conclusion, the test item is not classified as irritating, according to the CLP (EC 1272/2008) Regulation. The result is supported by the test on the analogous of the substance sodium salt.

EYE IRRITATION

The eye irritation potential was assessed following the procedure of the patch-test technique 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).

The primary irritation index in both rinsed and unrinsed eyes was estimated to be 0.

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 (30 seconds in the current study and 1 hours 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 for both rinsed and unrinsed eyes were 0 in all animals for all the reactions scored.

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

Justification for selection of skin irritation / corrosion endpoint:
Test conducted according to internationally accepted testing guidelines.

Justification for selection of eye irritation endpoint:
Test conducted according to internationally accepted testing guidelines

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 all the studies available, 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 EC 1272/2008 (CLP), 3.3 Serious eye damage/eye irritation section, substances which 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 for both rinsed and unrinsed eyes were 0 in all animals for all the reactions scored.

In conclusion, the available experimental data are adequate for classification and labelling and the substance is not classified for the eye and skin irritation, according to the CLP Regulation (EC 1272/2008).