Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-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

Study period:

From December 14 to 21, 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines.

Qualifier:

according to guideline

Guideline:

EPA OPP 81-5 (Acute Dermal Irritation)

Qualifier:

equivalent or similar 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: IVANOVAS GmBH, Med. Versuchstierzuchten KG, D-7964 Kisslegg.
- Age at study initiation: adult, age not specified.
- Weight at study initiation: 2 to 3 Kg.
- Housing: individually in metal cages.
- Diet: standard rabbit food NAFAG, No. 814 Gossau SG, ad libitum.
- Water: ad libitum.
- Acclimation period: minimum 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Photoperiod: 12 hours light cycle day.

Type of coverage:

occlusive

Preparation of test site:

other: intact and abraded skin.

Vehicle:

other: polyethylene glycol (PEG 400) + Saline (70:30)

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g of test material
- Application: gauze patches of 2.5 x2.5 cm laden with the test material.

VEHICLE
- Concentratio: 50 %

Duration of treatment / exposure:

The dressings were removed after a 24 hour application.

Observation period:

7 days.

Number of animals:

3 male and 3 female.

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 with the help of a "SchröpfSchnäpper", Aesculap, Switzerland.
- Type of wrap if used: the patches were covered with an impermeable material and were fastened to the body of the rabbit with adhesive tape.

SCORING SYSTEM
Skin reactions were assessed on removal and after 48 and 72 hours and 4 and 7 days.
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 #1

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 1.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

ca. 0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0.66

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

The calculated primary irritation index was 1.5.

Other effects:

One animal died after 6 days. No symptoms had been observed previously.

Intact skin reactions

Animal/sex	Reaction	24 hrs	48 hrs	72 hrs	4 days	7 days	Mean 24, 48 and 72 hrs
144	Erythema	2	1	0	0	0	0.75
145	Erythema	2	1	1	1	0	1.25
146	Erythema	2	1	0	0	0	0.75
147	Erythema	1	1	1	0	/	0.75
148	Erythema	1	0	0	0	0	0.25
149	Erythema	0	0	0	0	0	0.00
144	Oedema	2	1	0	0	0	0.75
145	Oedema	1	1	0	0	0	0.50
146	Oedema	2	1	0	0	0	0.75
147	Oedema	0	0	0	0	/	0.00
148	Oedema	0	0	0	0	0	0.00
149	Oedema	0	0	0	0	0	0.00

Abraded skin reactions

Animal/sex	Reaction	24 hrs	48 hrs	72 hrs	4 days	7 days	Mean 24, 48 and 72 hrs
144	Erythema	2	1	1	0	0	1.00
145	Erythema	2	2	1	1	0	1.50
146	Erythema	2	1	0	0	0	0.75
147	Erythema	2	2	1	1	/	1.50
148	Erythema	1	0	0	0	0	0.25
149	Erythema	1	0	0	0	0	0.25
144	Oedema	2	1	0	0	0	0.75
145	Oedema	1	0	0	0	0	0.25
146	Oedema	1	1	0	0	0	0.50
147	Oedema	2	1	1	1	/	1.25
148	Oedema	0	0	0	0	0	0.00
149	Oedema	0	0	0	0	0	0.00

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 tested in New Zealand White rabbits. The procedure used is described in the Proposed Guidelines of the United States Environmental Agency (EPA) § 163.81-5 "Primary dermal irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

Results

Under the conditions of the present experiment the test compound was found to cause a slight irritation when applied to intact and abraded rabbit skin. The calculated primary irritation index was 1.5. One animal died after 6 days.

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 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).

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)

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:

From December 14 to 21, 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines

Qualifier:

equivalent or similar 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: IVANOVAS GmBH, Med. Versuchstierzuchten KG, D-7964 Kisslegg.
- Weight at study initiation: 2 to 3 Kg.
- Housing: individually in metal cages.
- Diet: standard rabbit food NAFAG, No. 814 Gossau SG, ad libitum.
- Water: ad libitum.
- Acclimation period: minimum 4 days
Only rabbits with normal ophthalmic findings were used for these tests.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Photoperiod: 12 hours light cycle day.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 g

Duration of treatment / exposure:

single exposure; in 3 of the 6 rabbits approximately 30 seconds after the treatment the treated eye was washed out.

Observation period (in vivo):

7 days.

Number of animals or in vitro replicates:

3 females and 3 males.

Details on study design:

APPLICATION
The test material was inserted in the conjunctival sac of the left eye of the rabbits and the lids were gently closed for 15 seconds. The right eye was not treated and served as an untreated control.

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

TOOL USED TO ASSESS SCORE: hand-slit lamp.

SCORING SYSTEM
The eye irritation was assessed at 24, 48, 72 hours and 4 and 7 days after treatment.
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

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0.67

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

ca. 0.33

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

The primary irritation index was found to be: unrinsed eyes = 0.4; rinsed eyes = 0.1.

Other effects:

One animal died 7 days after instillation of the test material and it was stated that no symptoms had been observed prior to death.

Eye reactions

Animal	Eye	24 hrs	48 hrs	72 hrs	4 days	7 days	Mean 24, 48, 72 hrs
Cornea
144	not rinsed	0	0	0	0	0	0.00
145	not rinsed	0	0	0	0	0	0.00
146	not rinsed	0	0	0	0	0	0.00
147	rinsed	0	0	0	0	/	0.00
148	rinsed	0	0	0	0	0	0.00
149	rinsed	0	0	0	0	0	0.00
Iritis
144	not rinsed	0	0	0	0	0	0.00
145	not rinsed	0	0	0	0	0	0.00
146	not rinsed	0	0	0	0	0	0.00
147	rinsed	0	0	0	0	/	0.00
148	rinsed	0	0	0	0	0	0.00
149	rinsed	0	0	0	0	0	0.00
Conjunctival Redness
144	not rinsed	2	0	0	0	0	0.67
145	not rinsed	0	0	0	0	0	0.00
146	not rinsed	0	0	0	0	0	0.00
147	rinsed	1	0	0	0	/	0.33
148	rinsed	0	0	0	0	0	0.00
149	rinsed	0	0	0	0	0	0.00
Conjunctival Chemosis
144	not rinsed	0	0	0	0	0	0.00
145	not rinsed	0	0	0	0	0	0.00
146	not rinsed	0	0	0	0	0	0.00
147	rinsed	0	0	0	0	/	0.00
148	rinsed	0	0	0	0	0	0.00
149	rinsed	0	0	0	0	0	0.00

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 test was performed on 3 male and 3 female New Zealand White rabbits. The test material in an amount of 0.1 g was inserted into

the conjunctival sac of the left eye of the rabbits and the lids were gently closed for a few seconds. 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.

The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7 and was scored for each individual rabbit.

Results

Results

Under the conditions of this experiment the test material was found to cause a minimal irritation when applied to the rabbit eye mucosa.

The primary irritation index was found to be: unrinsed eyes = 0.4; rinsed eyes = 0.1.

Animal No. 147 died at day 7. No symptoms had been observed previously.

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 lower than 1 for corneal opacity, lower than 1 for irtitis, 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 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

The skin irritation potential was tested in New Zealand White rabbits. The procedure used is described in the Proposed Guidelines of the United States Environmental Agency (EPA) § 163.81-5 "Primary dermal irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978. Under the conditions of the experiment the test compound was found to cause a slight irritation when applied to intact and abraded rabbit skin. The calculated primary irritation index was 1.5. One animal died after 6 days.

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).

The eye irritation potential was tested on 3 male and 3 female New Zealand White rabbits. The test material in an amount of 0.1 g was inserted into the conjunctival sac of the left eye of the rabbits and the lids were gently closed for a few seconds. 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. Under the conditions of this experiment the test material was found to cause a minimal irritation when applied to the rabbit eye mucosa.

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 (1272/2008).

The mean values from gradings at 24, 48 and 72 hours for both rinsed and unrinsed eyes were lower than 1 for corneal opacity, lower than 1 for irtitis, lower than 2 for both conjunctival redness and oedema, in all animals.

Furthermore, two summaries reporting some toxicological test results is available on CAS 16470-24-9 (main component: 68 %; ramaining composition: sodium chloride, sodium sulphate and water). Unfortunately the original study reports cannot be more consulted. The available information included in the two summaries agree to indicate the substance as not eye irritating (Sandoz, 1972 and Sandoz 1974); only occasional, minor irritations were indicated for both intact and abraded skin (Sandoz, 1974).

REFERENCE

Sandoz (1972). Toxicologische Prüfung eines optischen Aufhellers. Unpublished data. Testing laboratory: Sandoz Agroforschung. Owner company: Archroma GmbH. Report N. 31/72. Report date: 1972-08-28.

Sandoz (1974). Toxicologische Prüfung eines optischen Aufhellers. Unpublished data. Testing laboratory: Sandoz Agro-department. Owner company: Archroma GmbH. Report N. 219/74. Report date: 1974-10-28.

Justification for selection of skin irritation / corrosion endpoint:

Study conducted according to internationally accepted testing guidelines

Justification for selection of eye irritation endpoint:

Study 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.

The 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 for both rinsed and unrinsed eyes were lower than 1 for corneal opacity, lower than 1 for irtitis, lower than 2 for both conjunctival redness and oedema, in all animals.

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