Reaction mass of [5-methyl-2-[[4-(4-methylanilino)-9,10-dioxo-1-anthryl]amino]phenyl]sulfonyloxypotassium and [5-methyl-2-[[4-(4-methyl-2-potassiooxysulfonyl-anilino)-9,10-dioxo-1-anthryl]amino]phenyl]sulfonyloxypotassium

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 20 to 23 November, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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 reversibke 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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1981

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Nottingham University, School of Agriculture, Sutton Bonington, Leicestershire, U.K.
- Age at study initiation: 12 to 16 weeks old.
- Weight at study initiation: 2.36 - 2.83 kg.
- Housing: rabbits were individually housed in suspected metal cages.
- Diet: rabbit diet, A.W. Tindall Ltd, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: minimum 5 days.
- Health check: within 24 hours of commencement of the test both eyes of each rabbit were examined for evidence of ocular irritation or defect using an ophthalmoscope. Animals showing evidence of ocular lesions were rejected and replaced.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 20 °C
- Relative humidity: 45 - 50 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: lighting was controlled by a time switch to give 12 hours light and 12 hours of darkness.

Vehicle:

unchanged (no vehicle)

Remarks:

test item was used as supplied

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml (weight 54 mg) of test item was placed into the right eye of each rabbit.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three animals

Details on study design:

TOOL USED TO ASSESS SCORE: examination was facilitated by use of a standard ophthalmoscope.

SCORING SYSTEM
Assessment of damage/irritation was made 1, 24, 48 and 72 hours following treatment, according to the numerical evaluation given by 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".

Cornea
Opacity: degree of density (area most dense taken for reading)
No opacity: 0
Scattered or diffuse area of opacity (other than slight dulling of normal lustre) ditails of iris clearly visible: 1
Easily discernible translucent area, details of iris slightly obscured: 2
Opalescent areas, no details of iris visible, size of pupil barely discernible: 3
Opaque cornea, iris not discernible through the opacity: 4

IRITIS
Normal: 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any 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

CONJUNCTIVAE
Redness (refers to lids and bulbar conjunctivae excluding cornea and iris)
Blood vessels normal: 0
Some blood vessels definitely hyperaemic (injected): 1
Diffuse, crimson colour, individual vessels not easily discernible: 2
Diffuse beefy red: 3

Chemosis: lids and/or nictitating membranes.
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

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

Reversibility:

fully reversible within: 24 hrs

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 48 hrs

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No evidence of corneal opacity was noted during the study period. Iridial inflammation was noted in all treated eye one hour after treatment. Minimal conjunctivitis continued to be noted on day one in one treated eye; the remaining two treated eyes were normal. All treated eyes were normal on day two.

Individual and mean scores for cornea, irirs and conjuntivae

Animal	Reaction	1 hrs	24 hrs	48 hrs	72 hrs	Mean 24/48/72 hrs
Animal 1	Cornea	0	0	0	0	0.0
Animal 2	Cornea	0	0	0	0	0.0
Animal 3	Cornea	0	0	0	0	0.0
Animal 1	Iris	0	0	0	0	0.0
Animal 2	Iris	1	0	0	0	0.0
Animal 3	Iris	1	0	0	0	0.0
Animal 1	Conjunctival redness	1S	1	0	0	0.3
Animal 2	Conjunctival redness	1S	0	0	0	0.0
Animal 3	Conjunctival redness	2S	0	0	0	0.0
Animal 1	Conjunctival chemosis	1	0	0	0	0.0
Animal 2	Conjunctival chemosis	1	0	0	0	0.0
Animal 3	Conjunctival chemosis	2	0	0	0	0.0

S: areas of green staining

Interpretation of results:

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

Conclusions:

Not 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 ml (weight 54 mg) 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 and 72 hours. The ocular reactions were scored by the method described by 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".

No evidence of corneal opacity was noted during the study period. Iridial inflammation was noted in all treated eye one hour after treatment. Minimal conjunctivitis continued to be noted on day one in one treated eye; the remaining two treated eyes were normal. All treated eyes were normal on day two.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

A sheet reporting the test results of an old experiment conducted on the test item is available; however, due to the fact that details on testing procedures and results are lacking, a reliability cannot be assiged. The result is here mentioned for completeness sake. The substance was indicated as not skin irritating, according to ETAD score system (reference 1976).

In order to assess the skin irritation potential toxicity of Acid Green 025, the available information on the structural analogous Similar Substance 02 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

The study conducted with Similar Substance 02, was performed in New Zealand White rabbit; the method used followed that described in the OECD Guidelines 404. 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.

EYE IRRITATION

The eye irritation potential of Acid Green 025 was investigated according to thhe method and procedures outlined into the OECD guideline 405. 0.1 ml (weight 54 mg) of test item was placed into the right eye of each rabbit. The ocular reactions were scored by the method described by 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". No evidence of corneal opacity was noted during the study period. Iridial inflammation was noted in all treated eye one hour after treatment. Minimal conjunctivitis continued to be noted on day one in one treated eye; the remaining two treated eyes were normal. All treated eyes were normal on day two.

A sheet reporting the test results of an old experiment conducted on the test item is available; however, due to the fact that details on testing procedures and results are lacking, a reliability cannot be assiged. The result is here mentioned for completeness sake. The substance was indicated as slightly eye irritating, according to ETAD score system (reference 1976).

In addition, the available data on structural analogous Similar Substance 01 have also been taken into consideration to comfirm the study outcomes. The read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

Three rabbits were used to test the eye irritation potential of the Similar Substance 01, in accordance with the OECD guideline 405. 0.1 g of the undiluted test material were placed into the conjunctival sac. Under the conditions of the experiment, the test item was found to cause a primary irritation score of 1.9 when applied to the rabbit eye mucosa.

Corneal opacity was observed in two animals duning the test. Anlmal no. 458 (male) showed sllght opacity from 1 to 48 hours, anirnal no. 459 (female) showed diffuse opacity from 1 to 24 hours after test article 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 chemosis, in all the tested animals. all the reactions were fully reversible within 72 hours.

REFERENCE

See attachment.

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 reversibke within 72 hours.

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.

In all the available experiments, 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 animals tested.

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