Disodium 3-[[4-amino-9,10-dihydro-9,10-dioxo-3-[sulphonato-4-(1,1,3,3-tetramethylbutyl)phenoxy]-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Endpoint summary

Description of key information

Not skin irritating

Eye Irrit. 2 (H319), according to the CLP Regulation (EC) No 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:

November from 20 to 23, 1990

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:

1981

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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, U.K.
- Age at study initiation: approximately twelve to sixteen weeks old.
- Weight at study initiation: 2.19 - 2.26 kg
- Housing: animals were individually housed in suspended metal cages.
- Diet: Spillers Rabbit Diet (Dalgety Agriculture Ltd., Almondsbury, Bristol) ad libitum.
- Water: mains drinking water, ad libitum.
- Acclimation period: a minimum acclimatisation period of five days.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 21 °C
- Humidity: 46 - 52 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The test material was moistened with distilled water immediately before application: 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:

1 male and 2 females

Details on study design:

TEST SITE
- Area of exposure: on the day before the test, each of a group 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. Test item was introduced under a 2.5 cm x 2.5 cm gauze 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 (BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing: any residual test material was removed by gentle swabbing with cotton wool soaked in distilled water followed by diethyl ether.
- Time after start of exposure: four hours after application the corset and patches were removed.

OBSERVATION TIME POINTS
Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined.

SCORING SYSTEM
The test sites were examined for evidence of primary irritation and scored according to the following scale from Draize J.H. (1959) Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics".

Erythema and eschar formation:
No eryrhema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Very slight oedema (barely perceptible) 1
Well-defined oedema (area well defined by definite raising) 2
Moderate oedema (raised approximately 1 millimeter) 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) 4

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 6 to give the primary irritation index of the test material. The test material was classified according to the following scheme:
Non-irritant, primary irritation index 0
Mild irritant, primary irritation index >0 - 2
Moderate irritant > 2 - 5
Severe irritant > 5 - 8

Irritation parameter:

erythema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2.3

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

edema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2.3

Reversibility:

fully reversible within: 48 hrs

Irritant / corrosive response data:

Faint purple-coloured staining caused by the test material was commonly noted during the study. This did not prevent accurate evaluation of skin responses.
Very slight erythema was noted at two treated skin sites one hour after patch removal, at all treated skin sites at the 24-hour observation and at two treated skin sites at the 48-hour observation.
Very slight oedema was confined to two treated skin sites at the 24-hour observation.
No skin irritation was noted 72 hours after treatment.

Individual scores

Animal	Skin Reaction	After				Mean 24/48/72 hrs
		1 hr	24 hrs	48 hrs	72 hrs
171 F	Erythema	0 STA	1 STA	1 STA	0 STA	0.67
172 M	Erythema	1 STA	1 STA	0 STA	0 STA	0.33
175 F	Erythema	1 STA	1 STA	1 STA	0 STA	0.67
171 F	Oedema	0	1	0	0	0.33
172 M	Oedema	0	0	0	0	0.00
175 F	Oedema	0	1	0	0	0.33

Sum of 24 and 72 hours reading: 5

Primary irritation index: 0.8, mild irritant

Interpretation of results:

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

Conclusions:

Mild irritant

Executive summary:

A study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 404 'Acute Dermal Irritation/Comosion' referenced as Method B4 in Commission Directive 84/449/EEC (which constitutes Annex V of Council Di recti ve 67/548/EEC).

A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced very slight erythema and very slight oedema. No skin irritation was noted 72 hours after treatment. The test material produced a primary irritation index of 0.8 and was classified as a mild 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. All the reactions were fully reversible within 72 hours.

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:

March from 13 to 20, 1984

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:

May 12, 1981

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland.
- Age at study initiation: 14 - 15 weeks.
- Weight at study initiation: 2.1 - 2.5 kg
- Housing: individually in stainless steel cages equipped with an automatic cleaning and drinking system.
- Diet: pelleted standand Kliba 341, Batch 94/84 rabbit maintenance diet ("Kliba", Kringentalmuehle AG, Switzerland), ad Iibitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation period: four days under laboratory conditions after veterinary exarnination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent Iight/12 hours dark.
- Other: at least 8 hours music/Iight period.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The individual dose volume was 0.1 g per animal.

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

1 male and 2 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: eyes were not rinsed.

OBSERVATIONS
Viability / Mortality: daily.
Body weights: pre-test, day 1 and at termination of test on a Mettler PK 16 balance.

TOOL USED TO ASSESS SCORE: eye examinations were made with a slit-Iamp 30 SL (c. Zeiss AG, Zuerich / Switzerland) and a Varta Cliptrix diagnostic-Iamp (A. Riegger, BaseI / Switzerland).

SCORING SYSTEM
The eye of each animal were examined 1-, 24-, 48-, 72 hours and 7 days after administration.
The irritation was assessed according to the OECD Guidelines for testing of Chemicals, Section 4, number 405 "Acute Eye irritation/corrosion " adopted May 12, 1981. The corrosive properties of the test article the treated and the color of eye were described and recorded.

Corneal irritation
Opacity degree of density (denest area used for assessment)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than sIight dulling of normal luster), details of iris clearly visible 1
EasiIy discernible translucent area, details of iris slightly obscured 2
Nacrous area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 4

Iridic irritation
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination thereof, iris still reacting to Iight (sluggish reaction is positive) 1
No reaction to light, hemorrhage, gross destruction (any or aII of these) 2

Conjunctival irritation
Redness (refers to palpebrar and bulbar conjunctivae, cornea and iris)
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse, crimson coIor, individual vessels not easily discernible 2
Diffuse beefy red 3

Chemosis, lids and/or nictating membranes
No sweIIing 0
Any swelling above normal (includes nictating membranes) 1
Obvious swelling with partial eversion of lids 2
SweIIing with Iids about half closed 3
SweIIing with Iids more than half closed 4

Irritation parameter:

cornea opacity score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

1.3 - 1.7

Irritation parameter:

iris score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

1

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

2 - 3

Irritation parameter:

chemosis score

Basis:

animal: 2/3

Time point:

24/48/72 h

Score:

< 2

Irritant / corrosive response data:

Test item showed a primary irritation score of 5.6 when applied to the rabbit eye mucosa.
In the area of application a violet discoloration of the corneal, scIera and conjunctivae was observed within 1 hour after treatment. This could be related to effects of the test article.
No corrosion of the cornea was observed at each of the measuring intervals.

INDIVIDUAL FINDINGS
Animal No 326, male
1 hour - Cornea: opacity, whole, discoloured, ventral. Conjunctivae: edema, clear visible; discoloured, severe, including nictating membrane and conjunctival sac; substance remainders, violet mucous. Sclera: not visible. Discharge: severe.
24 hours - Cornea: opacity, whole, discoloured, ventral. Iris: slight reddened. Conjunctivae: reddened, severe; edema severe. Sclera: not visible. Discharge: severe.
48 hours - Cornea: opacity, severe. Iris: slight reddened. Conjunctivae: reddened, severe; edema severe. Sclera: not visible. Discharge: severe.
72 hours - Cornea: opacity, severe. Conjunctivae: reddened, severe; edema severe. Sclera: not visible. Discharge: severe.
7 days - Cornea: opacity, ventral. Iris: reddened, ventral, slight. Conjunctivae: reddened. Discharge: minimal.

Animal No 327, female
1 hour - Cornea: opacity, whole, discoloured, ventral. Conjunctivae: edema, clear visible; discoloured, severe, including nictating membrane and conjunctival sac; substance remainders, violet mucous. Sclera: not visible. Discharge: severe.
24 hours - Cornea: opacity, whole. Iris: reddened. Conjunctivae: reddened; edema. Sclera: reddened. Discharge: severe.
48 hours - Cornea: opacity, whole. Conjunctivae: reddened; edema. Sclera: reddened. Discharge: severe.
72 hours - Cornea: opacity, slight, diffuse. Conjunctivae: reddened; edema slight. Sclera: reddened. Discharge: moderate.
7 days - Cornea: opacity, clear visible, marginal area. Conjunctivae: reddened, slight.

Animal No 328, female
1 hour - Cornea: reddened, slight; edema, clear visible; discoloured including nictating membrane; substance remainders, violet mucous. Discharge: severe.
24 hours - Cornea: opacity, whole. Iris: reddened. Conjunctivae: reddened; edema. Sclera: reddened. Discharge: severe.
48 hours - Cornea: opacity. Iris: reddened, slight. Conjunctivae: reddened, clear visible; edema, slight. Sclera: reddened. Discharge: severe.
72 hours - Cornea: opacity. Iris: reddened, slight. Conjunctivae: reddened, clear visible; edema slight. Sclera: reddened. Discharge: minimal.
7 days - Cornea: opacity, ventral, minimal.

Other effects:

TOXIC SYMIPTONS / MORTALITY
No acute toxic symptoms were observed in the animals during the test period and no mortality occurred.

BODY WEIGHT
The body weight gain of all rabbits was similar.

Eye irritation score

Animal	Reaction	After					Mean 24/48/72 hrs
		1 hr	24 hrs	48 hrs	72 hrs	7 days
326 M	Cornea opacity	1	1	2	2	2	1.67
327 F	Cornea opacity	1	1	2	1	1	1.33
328 F	Cornea opacity	0	1	2	2	1	1.67
326 M	Iris	0	1	1	1	1	1.00
327 F	Iris	0	1	0	0	0	0.33
328 F	Iris	0	1	1	1	0	1.00
326 M	Conjunctival redness	0	3	3	3	2	3.00
327 F	Conjunctival redness	0	2	2	2	1	2.00
328 F	Conjunctival redness	1	2	2	2	0	2.00
326 M	Conjunctival chemosis	2	2	3	2	0	2.33
327 F	Conjunctival chemosis	3	2	2	1	0	1.67
328 F	Conjunctival chemosis	2	2	1	1	0	1.33

Interpretation of results:

other: Eye Irrit 2 (H319), according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Eye irritating

Executive summary:

The eye irritation potential of the test substance was investigated according to thhe method and procedures outlined into the OECD guideline 405. 0.1 g of test item was placed into the right eye of each rabbit; the other eye remaining untreated, served as a control. The eyes were examined and the grade of ocular reaction was recorded at 1, 24, 48, 72 hours and 7 days. The ocular reactions were scored by the method described into the OECD Guidelines for testing of Chemicals, Section 4, number 405 "Acute Eye irritation/corrosion" adopted May 12, 1981.

Under the conditions of the experiment, test item was found to cause a primary irritation score of 5.6 when applied to the rabbit eye mucosa. In the area of application, a violet discoloration sclera and conjunctivae was observed within 1 hour after treatment. This could be related to effect of the test article. No corrosion was observed at each of the measuring intervals.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were higher than 1, but lower than 3 for corneal opacity (i.e. 1 .3 - 1.7) in all the treated animals; equal to 1 for iritis in 2 out of 3 rabbits, equal to 2 in two rabbits and equal to 3 in the third one for conjunctival redness; lower than 2 in 2 out of 3 tested rabbits for conjunctival oedema. Although the experiment was termined after 7 days and some reactions were still visible, there are no resons to suspect the possibility of non-reversibility of the effects over an observation period of normally 21 days: based on the description of the individual findings provided in the report, the severity of the effects resulted to be reduced respect to severity recorded after 72 hours.

In conclusion, a classification in category 2 of CLP Regulation (EC) No 1272/2008 seems to be appropriate.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

SKIN IRRITATION

A study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method used followed that described in the OECD Guidelines (1981) No. 404 referenced as Method B4 in Commission Directive 84/449/EEC.

A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced very slight erythema and very slight oedema. No skin irritation was noted 72 hours after treatment. The test material produced a primary irritation index of 0.8 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

In addition, an old test is available. The procedure employed was that prescribed by The Food and Drug Administration of the U.S.A. in The Federal Register (17 September 1964 191.11). Verv slight erythema only was observed in one intact and four abraded sites ot the 24 hours-reading. Very slight oedema,only was observed in one intact and one abraded site. Very slight erythema with or without very stight oedema was observed in three abraded sites only at the 72 hours reading. One animal did not show any obserable response to treatment throughout the 72 hours observation period.

Unfortunately, the reliability of the data cannot be judged due to the lacking of details about the actual tested lot composition.

EYE IRRITATION

The eye irritation potential of the test substance was investigated according to the method and procedures outlined into the OECD guideline 405. 0.1 g of test item was placed into the right eye of each rabbit; the other eye remaining untreated, served as a control. The eyes were examined and the grade of ocular reaction was recorded at 1, 24, 48, 72 hours and 7 days.

Under the conditions of the experiment, test item was found to cause a primary irritation score of 5.6 when applied to the rabbit eye mucosa. In the area of application, a violet discoloration sclera and conjunctivae was observed within 1 hour after treatment. This could be related to effect of the test article. No corrosion was observed at each of the measuring intervals.

In addition, an old test is available. The procedure employed was that prescribed by The Food and Drug Administration of the U.S.A. in The Federal Register (17 September 1964 191.12). Test item was instilled into the eyes of three rabbits without rinsing and the eyes of the three remaining rabbits were irrigated with 20 ml of lukewarm water 5 seconds after instillation.

All three animals, of which eyes were not rinsed, gave "positive" reactions: corneal opacities developed in all three animals and were still present, with pannus formation, in two animals on day 14. Staining of the cornea by the dye was observed in two animals. Considerable swelling of the conjunctivae with partial eversion of the eyelids, or with the eyelids about half closed, was observed in all three animals. This was accompanied in one animal, by a diffuse crimson-red colouration of the conjunctivae.

Also animals, of which eyes were rinsed after 5 seconds gave a "positive" reaction: a corneal opacity was observed in one animal; temporary dulling of the normal corneal lustre was observed in the other two animals. Staining of the cornea by the dye was observed in all three animals. Considerable swelling of the conjunctivae with partial eversion of the eyelids was observed in one animal. Temporary mild conjunctival reactions were observed in the two remaining animals.

Unfortunately, the reliability of the data cannot be judged due to the lacking of details about the actual tested lot composition.

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. All the reactions were fully reversible within 72 hours.

Thus, the test item does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC) No 1272/2008.

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.

The mean values from gradings at 24, 48 and 72 hours were higher than 1, but lower than 3 for corneal opacity (i.e. 1 .3 - 1.7) in all the treated animals; equal to 1 for iritis in 2 out of 3 rabbits, equal to 2 in two rabbits and equal to 3 in the third one for conjunctival redness; lower than 2 in 2 out of 3 tested rabbits for conjunctival oedema. Although the experiment was termined after 7 days and some reactions were still visible, there are no resons to suspect the possibility of non-reversibility of the effects over an observation period of normally 21 days: based on the description of the individual findings provided in the report, the severity of the effects after 7 days resulted to be reduced respect to severity recorded after 72 hours.

Thus, a classification in category 2 of CLP Regulation (EC) No 1272/2008 seems to be appropriate.