Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Endpoint summary

Description of key information

Not skin irritating (not classified, according to the CLP Regulation (EC) 1272/2008)

Potentially mild eye irritating (not classified, according to the CLP Regulation (EC) 1272/2008)

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:

May from 09 to 12, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

For the purpose of the study the test material was used as supplied.
The pH of the test material was determined prior to commencement of the study and found to be 9.1 at 10 % w/w aqueous preparation of the test material.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd, Moston, Sandbach, Cheshire, UK.
- Weight at study initiation: 2.0 to 3.5 kg
- Housing: the animals were individually housed in suspended cages.
- Diet: ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 23 °C
- Humidity: 30 - 70 %
- Air changes: the rate of air exchange was at least fifteen changes per hour.
- Photoperiod: the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 g of the test material, moistened with 0.5 ml of distilled water

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

three rabbits

Details on study design:

TEST SITE
- Area of exposure: on the day before the test each of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
- Application: test material was introduced under a 2.5 cm x 2.5 cm cotton garze patch and placed in position on the shorn skin.
- Type of wrap if used: the patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing: any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours.

OBSERVATION TIME POINTS
Approximately one hour following the removal of the patches and 24,48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored.
The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary initation index of the test material. The test material was classified according to the scheme devised by Draize J H (1959) "Dermal Toxicity" In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the United States, Austin, Texas, p.47.

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 eschar formation preventing grading of erythema 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 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

Irritation parameter:

erythema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Yellow-coloured staining was noted at all treated skin sites throughout the study. This did not affect evaluation of skin reactions. No evidence of skin irritation was noted during the study.
The test material produced a primary irritation index of 0.0. No corrosive effects were noted.

Individual skin reactions

Animal	Skin Reaction	Time				Mean 24/48/72 hrs
		1 hr	24 hrs	48 hrs	72 hrs
Male 155	Erythema/Eschar	0 STA	0 STA	0 STA	0 STA	0 STA
Male 156	Erythema/Eschar	0 STA	0 STA	0 STA	0 STA	0 STA
Male 157	Erythema/Eschar	0 STA	0 STA	0 STA	0 STA	0 STA
Male 155	Oedema	0	0	0	0	0
Male 156	Oedema	0	0	0	0	0
Male 157	Oedema	0	0	0	0	0

STA : Yellow-coloured staining

Sum of 24 and 72-hour Readings (S): 0

Primary Irritation Index (8/6): 0/6 = 0.0

Classification: non-irritant

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Non skin irritating

Executive summary:

The study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" and Method B4 Acute Toxicity (Skin lrritation) of Commission Directive 2004/73/EC. A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced no evidence of skin irritation.

The test material produced a primary irritation index of 0.0 and was classified as non-irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Conclusion

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, thus the test item does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC) No 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

Study period:

May from 15 to 26, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test material was used as supplied.
The pH of the test material was determined prior to commencement of the study and found to be 9.1 at 10 % w/w aqueous preparation of the test material.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd, Moston, Sandbach, Cheshire, UK.
- Age at study initiation: twelve to twenty weeks old.
- Weight at study initiation: 2.0 to 3.5 kg
- Housing: the animals were individually housed in suspended cages.
- Diet: Certifred Rabbit Diet (Code 5322) supplied by BCM IPS Limited, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: at least 5 days.
- Health check: immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 23 °C
- Humidity: 30 - 70 %
- Air changes: the rate of air exchange was at least fifteen changes per hour.
- Photoperiod: the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A volume of 0.1 ml of the test material, which was found to weigh approximately 87 mg was placed into the conjunctival sac of the right eye.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three rabbits

Details on study design:

TESTING PROCEDURES
Initially, a single rabbit was treated. The test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material and then released.
Immediately after administration of the test material, an assessment of the initial pain reaction was made.
After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

TOOL USED TO ASSESS SCORE:examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

SCORING SYSTEM
'Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given by Draize J. H. (1977.
Any other ocular effects were also noted.
The numerical values corresponding to each animal, tissue and observation time were recorded.
The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated.
Using the numerical data obtained a modified version of the system described by Kay JH and Calandra JC (1962), J. Soc. Cosmet. Chem. 13, 281-289 was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the comea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material.
If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

Draize Scale for Scoring Ocular Irritation
CONJTINCTIVAE
(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal 0
Vessels definitely injected above normal 1
More diffirse, deeper crimson red, individual vessels not easily discernible 2
Diffiuse 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 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 a considerable a¡ea a¡ound the eye 3

THE TOTAL SCORE: (A + B + C) x 2
MAXIMUM TOTAL = 20

IRIS
(D) Values
Normal 0
Folds above normal, congestion, swelling, circumcomeal 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

THE TOTAL SCORE = D x 5
MAXIMIUM TOTAL = 10

CORNEA
(E) Degree of Opacity (most dense area used)
No opacity 0
Scattered or diffiuse areas, details of iris clearly visible 1
Easily discemible translucent areas, details of iris slightly obscured 2
Opalescent areas, no details ofiris visible, size ofpupil barely discernible 3
Opaque, iris not discernible through the opacity 4

(F) Area of Cornea Involved
One quarter (or less) but not zero 1
Greater than one quarter but less than half 2
Greater than halfbut less than three quarters 3
Greater than three quarters, up to whole area 4

THE TOTAL SCORE = (E x F) x 5
MAXIMIUM TOTAL = 80

MAXIMUM TOTAL SCORE POSSIBLE = 110

Irritation parameter:

cornea opacity score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 48 hrs

Irritant / corrosive response data:

Yellow-coloured staining of the fur was noted around all treated eyes throughout the study. No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one hour after treatment.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal to moderate conjunctival irritation noted in all treated eyes at the 24-hours observation and minimal conjunctival irritation noted in all treated eyes at the 48-hours observation.
All treated eyes appeared normal at the 72-hours observation.
The test material produced a maximum group mean score of 13.7 and was classified as a mild irritant to the rabbit eye according to a modified Kay and Calandra classification system.

Animal N.	Reaction	Observation at				Mean 24/48/72 hrs
		1 hr	24 hrs	48 hrs	72 hrs
64 Male	Corneal opacity	0	0	0	0	0.00
12 Male	Corneal opacity	0	0	0	0	0.00
14 Male	Corneal opacity	0	0	0	0	0.00
64 Male	Iris	1	0	0	0	0.00
12 Male	Iris	0	0	0	0	0.00
14 Male	Iris	0	0	0	0	0.00
64 Male	Conjunctival redness	2	2	1	0	1.00
12 Male	Conjunctival redness	2	1	1	0	0.67
14 Male	Conjunctival redness	2	1	1	0	0.67
64 Male	Conjunctival chemosis	2	1	0	0	0.33
12 Male	Conjunctival chemosis	2	1	0	0	0.33
14 Male	Conjunctival chemosis	2	1	0	0	0.33

Individual Scores and Individual Total Scores for Ocular Irritation

	64 Male				12 Male				14 Male
	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs
CORNEA
E: Degree of Opacity	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
IRIS
D	1	0	0	0	0	0	0	0	1	0	0	0
Score (D x 5)	5	0	0	0	0	0	0	0	5	0	0	0
CONJUNCTIVAE
A: Redness	2	2	1	0	2	1	1	0	2	1	1	0
B = Chemosis	2	1	0	0	2	1	0	0	2	1	0	0
C: Discharge	2Sf	1Sf	0Sf	0Sf	2Sf	1Sf	0Sf	0Sf	2Sf	1Sf	0Sf	0Sf
Score(A+B+C)x2	12	8	2	0	12	6	2	0	12	6	2	0
Total Score	17	8	2	0	12	6	2	0	12	6	2	0

Sf = Yellow-coloured staining ofthe fur a¡ound the treated eve

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Mild irritant.

Executive summary:

The study was performed to assess the iritancy potential of the test material to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the OECD Guidelines 405 and EU Method B5.

A single application of the test material to the non-irrigated eye of three rabbits produced iridial inflammation and moderate conjunctival irritation. All treated eyes appeared normal at the 72-hour observation.

The test material produced a maximum group mean score of 13.7 and was classified as a mild irritant to the rabbit eye according to a modified Kay and Calandra classification system.

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 the tested animals. Thus, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC) No 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 irritancy potential of the test material to the skin was investigated in New Zealand White rabbit. The method was designed according to the OECD Guidelines 404 and the EU Method B4. A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced no evidence of skin irritation. The test material produced a primary irritation index of 0.0 and was classified as non-irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

EYE IRRITATION

The irritancy potential of the test material to the eye was assayed using the New Zealand White rabbit. The method was designed to meet the requirements of the OECD Guidelines 405 and EU Method B5. A single application of the test material to the non-irrigated eye of three rabbits produced iridial inflammation and moderate conjunctival irritation. All treated eyes appeared normal at the 72-hour observation. The test material produced a maximum group mean score of 13.7 and was classified as a mild irritant to the rabbit eye according to a modified Kay and Calandra classification system.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 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, thus the test item does not meet the criteria to be classified as irritating.

According to the CLP Regulation (EC) No 1272/2008, serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application. Eye irritation means the production of changes in the eye, which are fully reversible within 21 days of application.

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 the tested animals. All the reactions resulted to be fully reversible within 72 hours, thus, the substance does not meet the criteria to be classified as eye irritating.

In conclusion, the substance is not classified for the eye/skin irritation, according to the CLP Regulation (EC) No. 1272/2008.