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Reaction mass of disodium 2-({4-methyl-2-[(4-{[5-methyl-1-(2-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)methyl]pentyl}amino)benzene-1-sulfonate,disodium 4-{5-methyl-3-[(4-{[5-methyl-1-(4-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]hexyl}benzene-1-sulfonate anddisodium 4-{5-methyl-3-[(4-{[5-methyl-1-(2-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]hexyl}benzene-1-sulfonate
EC number: 947-270-4 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 24 June 2003 to 24 July 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 003
- Report date:
- 2003
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.6 (Skin Sensitisation)
- Version / remarks:
- Commission Directive 96/54/EC Annex IV C Method B.6. Skin Sensitisation Adopted July 30, 1996
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- OECD Guidelines for Testing of Chemicals Section 4 Health Effects 406 Skin Sensitisation Adopted July 17, 1992
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.2600 (Skin Sensitisation)
- Version / remarks:
- U.S. EPA: OPPTS 870.2600, Health Effects Test Guidelines: Skin sensitization, August 1998
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of study:
- guinea pig maximisation test
- Justification for non-LLNA method:
- Study available is over 12 years old.
Test material
- Reference substance name:
- Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)
- EC Number:
- 276-822-0
- EC Name:
- Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)
- Cas Number:
- 72749-90-7
- Molecular formula:
- C40H46N2O8S2.2Na
- IUPAC Name:
- Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)
- Test material form:
- solid: particulate/powder
- Details on test material:
- Acid Blue 221
Constituent 1
- Specific details on test material used for the study:
- No further details specified in the study report.
In vivo test system
Test animals
- Species:
- guinea pig
- Strain:
- Dunkin-Hartley
- Sex:
- female
- Details on test animals and environmental conditions:
- Species of animals: Guinea Pig
Strain of animals: Mol:DH (Moellegaard)
Origin (supplier) of animals: M & B A/S, P.O.Box 1079, DK-8680 Ry, Denmark
Animal identification: fur marking with KMnO4 and cage numbering
Body weight of the main test group at start of study: mean = 342g (= 100 %); min = 290 g (- 15.2 %); max = 360 g (+ 4.8 %); n = 15
Randomization procedure: Computer generated algorithm (archived with raw data). Randomization schemes 2001.0644
HOUSING AND CARE OF ANIMALS
Animal maintenance: in transparent macrolon® cages (type V) on soft wood granulate in an air-conditioned room, 5 animals per cage
Room temperature: 22 ± 3 °C (except short lasting deviations due to disturbances of air condition)
Relative humidity: 50 ± 20 % (except short lasting deviations due to disturbances of air condition)
Lighting times: 12 hours light / dark cycle
Acclimatization: at least five days
Food: ssniff® Ms-H (V 2233), ad libitum
Water: tap water in plastic bottles, ad libitum
Study design: in vivo (non-LLNA)
Inductionopen allclose all
- Route:
- intradermal
- Vehicle:
- water
- Concentration / amount:
- 5%
- Day(s)/duration:
- one administration on day 1
- Adequacy of induction:
- highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
- Route:
- intradermal
- Vehicle:
- other: 50 % Freund’s Complete Adjuvant emulsion
- Concentration / amount:
- 5%
- Day(s)/duration:
- one administration on day 1
- Adequacy of induction:
- highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
- Route:
- epicutaneous, occlusive
- Vehicle:
- water
- Concentration / amount:
- 25%
- Day(s)/duration:
- Day 8 / 48 hours
- Adequacy of induction:
- other: No signs of irritation occurred after administration of the different test concentrations.
Challenge
- No.:
- #1
- Route:
- epicutaneous, occlusive
- Vehicle:
- water
- Concentration / amount:
- 25%
- Day(s)/duration:
- Day 22 for 24 hours
- Adequacy of challenge:
- highest non-irritant concentration
- No. of animals per dose:
- 10 animals in treatment group
5 animals in control group - Details on study design:
- TEST PROCEDURE
The following preparations were used for the intradermal injections
Control group
1.) 50 % Freund’s Complete Adjuvant emulsion
Original Freund’s Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
2.) deionized water (Vehicle)
3.) 50 % Freund’s Complete Adjuvant emulsion mixed with an equal volume of the vehicle
Treatment group
1.) 50 % Freund’s Complete Adjuvant emulsion
Original Freund’s Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
2.) 5 % Acid Blue 221 in deionized water
3.) 5 % Acid Blue 221 in a 50 % Freund’s Complete Adjuvant emulsion
For the intradermal injections of the test substance in 50 % Freund’s Adjuvant, Telon Blue MGLW was dissolved in deonized water and then mixed with an equal volume of Freund’s Original Adjuvant [percentages w/v].
For the dermal treatments, Telon Blue M-GLW was suspended in vehicle [percentages w/v].
DETERMINATION OF THE PRIMARY NON-IRRITATING CONCENTRATION
Prior to the determination of the primary non-irritating concentration in a dermal-occlusive test three animals each received 4 intradermal injections of a 50% Freund’s Complete Adjuvant emulsion (4 x 0.1 mL) into the dorsal area, since Freund’s Complete Adjuvant may lower the threshold of primary irritation. Thereafter, the test substance was administered to the flanks of the guinea pigs according to the following procedure: See Table in “Any other information”.
The hair on the flanks of the animals was removed mechanically. 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema according to the scale of Draize.
DETERMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 2 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders.
24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.
MAIN TEST FOR THE SENSITIZING PROPERTIES
Chronological description of the test procedure indicating the day, at which procedure was carried out, on the left margin of the page.
Study day -1
The body weight of the animals was determined.
The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.
Study day 1
Intradermal induction treatment.
Two intradermal injections per animal of the following preparations were applied. The injection sites (site 1, 2 and 3) were all within a dorsal area of 2 x 4 cm. The injection sites were left uncovered.
Study days 2 - 7
The administration area was examined for local tolerance. Systemic toxic effects were recorded, when apparent.
Study day 8
Dermal induction treatment.
An amount of 0.5 mL of the test substance preparation (treatment group) or the deionized water (control group) was administered to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept under an occlusive bandage covered with an impermeable film and an elastic bandage for 48 hours.
Treatment group: 25 % test substance in deionized water
Control group: deionized water
Study day 10
Occlusive bandage was removed, irritant effects were recorded, when apparent.
Study days 11 - 21
No treatment of control or treatment group.
Test animals were kept under observation.
Study days 22
Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.
An amount of 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 hours under an occlusive bandage covered with an impermeable film and an elastic bandage.
Treatment and control group (left flank): 25 % Telon Blue M-GLW in deionized water
Study day 23
Occlusive bandage was removed. Any remnants of the test substance were carefully washed off with warm water.
Study day 24
Examination of the skin approx. 24 hours after removal of the patches.
Study day 25
Examination of the skin approx. 48 hours after removal of the patches.
Body weight of the test animals was determined. - Positive control substance(s):
- yes
- Remarks:
- Alpha-Hexylcinnamaldehyde
Results and discussion
- Positive control results:
- After the challenge treatment nine animals of the treatment group (90%) showed a positive reaction during the observation period.
In vivo (non-LLNA)
Resultsopen allclose all
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- 25%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Clinical observations:
- no clinical signs of intoxication throughout the study.
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- 25%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Clinical observations:
- no clinical signs of intoxication throughout the study.
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- 25%
- No. with + reactions:
- 0
- Total no. in group:
- 5
- Clinical observations:
- After the challenge treatment no animals of the treatment group showed a positive skin reaction during the observation period.
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- 25%
- No. with + reactions:
- 0
- Total no. in group:
- 5
- Clinical observations:
- After the challenge treatment no animals of the treatment group showed a positive skin reaction during the observation period.
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- positive control
- Dose level:
- 25 %
- No. with + reactions:
- 9
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- positive control
- Dose level:
- 25 %
- No. with + reactions:
- 9
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
Any other information on results incl. tables
Bodyweights – Individual Bodyweights
Bodyweights (g) |
|||
Day numbers relative to Start Date |
|||
Group Sex |
Animal Number |
-1 |
25 |
1f |
1 2 3 4 5 |
343 308 315 368 340 |
507 432 474 511 488 |
|
Mean S.D. N |
334.8 24.0 5 |
482.4 31.9 5 |
* = result to left has an associated comment or marker
Nominal Dose: Group 1 – Control Group Group 2 – Treatment Group
Bodyweights (g) |
|||
Day numbers relative to Start Date |
|||
Group Sex |
Animal Number |
-1 |
25 |
2f |
6 7 8 9 10 11 12 13 14 15 |
352 366 343 290 330 360 360 306 372 375 |
528 537 494 438 484 513 485 456 553 540 |
|
Mean S.D. N |
345.4 28.6 10 |
502.38 37.9 10 |
* = result to left has an associated comment or marker
Nominal Dose: Group 1 – Control Group Group 2 – Treatment Group
Bodyweight Gains – Individual Bodyweight Gains
g |
|||
Day numbers relative to Start Date |
|||
Group Sex |
Animal Number |
From: To: |
-1 25 |
1f |
1 2 3 4 5 |
|
164 124 159 143 148 |
|
Mean: S.D. N |
147.6 15.6 5 |
* = result to left has an associated comment or marker
Nominal Dose: Group 1 – Control Group Group 2 – Treatment Group
g |
|||
Day numbers relative to Start Date |
|||
Group Sex |
Animal Number |
From: To: |
-1 25 |
2f |
6 7 8 9 10 11 12 13 14 15 |
|
176 171 151 148 154 153 125 150 181 165 |
|
Mean: S.D. N |
157.4 16.4 10 |
* = result to left has an associated comment or marker
Nominal Dose: Group 1 – Control Group Group 2 – Treatment Group
DERMAL OBSERVATION
Determination of the tolerance of the intradermal injections – summary, 2 animals
Tolerance of intradermal injections – individual values
Time after injection |
Findings |
||
5.0% |
1.0% |
0.2% |
|
Day 1 |
Clear edema * |
Slight edema * |
Slight edema * |
Day 2 |
Clear edema * |
Slight edema * |
Slight edema * |
Day 3 |
Clear edema * |
Slight edema * |
Slight edema * |
Day 4 |
Slight edema * |
NAD |
NAD |
*Due to the color of the test substance the treated skin of the animals could not be assessed for erythema.
Determination of the primary non-irritating concentration – individual values
Treated area: left and right flank
Time of observation: approx.. 24 hours after removal of the patches
Non-irritating test – individual values according to the score of Draize
Animal No. |
Concentration [%] |
Results |
|
1 left flank |
25 |
Erythema Edema |
0 0 |
1 right flank |
5 |
Erythema Edema |
0 0 |
2 left flank |
25 |
Erythema Edema |
0 0 |
2 right flank |
5 |
Erythema Edema |
0 0 |
3 left flank |
25 |
Erythema Edema |
0 0 |
3 right flank |
5 |
Erythema Edema |
0 0 |
Observation of skin during intradermal induction treatment
Control group (5 animals)
Side 1: 50% Freund’s Adjuvant
Side 2: deionized water
Side 3: equal volume of deionised water and 50% Freund’s Adjuvant
Intradermal induction – individual values control group
Study day |
Findings |
||
Side 1 |
Side 2 |
Side 3 |
|
2 |
Severe erythema and edema |
No findings |
Severe erythema and edema |
3 |
Severe erythema and edema |
No findings |
Severe erythema and edema |
4 |
Severe erythema and edema |
No findings |
Severe erythema and edema |
5 |
Severe erythema and edema sporadically encrusted and indurated |
No findings |
Severe erythema and edema sporadically encrusted and indurated |
6 |
Severe erythema and edema sporadically encrusted and indurated |
No findings |
Severe erythema and edema sporadically encrusted and indurated |
7 |
Still clear signs of irritation, hence, 10% sodium dodecylsulfate was not administered |
Treatment group (10 animals)
Side 1: 50% Freund’s Adjuvant
Side 2: 5% Telon Blue M-GLW
Side 3: 5% Telon Blue M-GLW in 50% Freund’s Adjuvant
Intradermal induction – individual values treatment group
Study day |
Findings |
||
Side 1 |
Side 2 |
Side 3 |
|
2 |
Severe erythema and edema |
Clear edema * |
Severe edema * |
3 |
Severe erythema and edema |
Clear edema * |
Severe edema * |
4 |
Severe erythema and edema |
Clear edema * |
Severe edema * |
5 |
Severe erythema and edema, sporadically encrusted, indurated |
Clear edema * |
Severe edema *, sporadically encrusted, indurated |
6 |
Severe erythema and edema, sporadically encrusted, indurated |
Clear edema * |
Severe edema *, sporadically encrusted, indurated |
7 |
Still clear signs if irritation, hence, 10% sodium dodecysulfate was not administered. |
*Due to the color of the test substance the treated skin of animals could not be assessed for erythema.
Observation of skin irritation during dermal induction treatment
Treatment
Control group: deionized water (day 8)
Treatment group: 25% Telon Blue M-GLW in deionized water (day 8)
Time of observation: approx.. 48 hours after administration (day 10)
Dermal induction – individual values control group
Findings |
||
Site 1 (50% FCA) |
Site 2 (vehicle) |
Site 3 (equal volume of vehicle and 50% FCA) |
Severe erythema and edema, sporadically encrusted, indurated, necrosis |
No findings |
Severe erythema and edema, sporadically encrusted, indurated, necrosis |
Dermal induction – individual values treatment group
Findings |
||
Site 1 (50% FCA) |
Site 2 (5%) |
Site 3 (5% in 50% FCA) |
Severe erythema and edema, sporadically encrusted, indurated, necrosis |
Clear edema, sporadically encrusted, indurated, necrosis * |
Severe edema, sporadically encrusted, indurated, necrosis * |
*Due to the color of the test substance the treated skin of the animals could not be assessed for erythema.
Challenge treatment – control and treatment group
25% Telon Blue M-GLW in deionized water (day 22)
Treated area: left flank
Time of observation: approx.. 24 hours removal of the patches (day 24)
Challenge treatment – individual values (day 24)
Control animal No.: |
1 |
2 |
3 |
4 |
5 |
|
|
|
|
|
Value |
0 |
0 |
0 |
0 |
0 |
|
|
|
|
|
Treated animal No.: |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
Value |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Time of observation: approx.. 48 hours removal of the patches (day 25)
Challenge treatment – individual values (day 25)
Control animal No.: |
1 |
2 |
3 |
4 |
5 |
|
|
|
|
|
Value |
0 |
0 |
0 |
0 |
0 |
|
|
|
|
|
Treated animal No.: |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
Value |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
After the challenge treatment no animals of the treatment group showed a positive skin reaction during the observation period.
POSITIVE CONTROL ASSAY
Challenge treatment – control and treatment group
25% Alpha-Hexylcinnamaldehyde in polyethylene glycole 400 (day 22)
Treated area: left flank
Time of observation: approx.. 24 hours removal of the patches (day 24)
Challenge treatment – individual values (day 24)
Control animal No.: |
1 |
2 |
3 |
4 |
5 |
|
|
|
|
|
Value |
0 |
0 |
0 |
0 |
0 |
|
|
|
|
|
Treated animal No.: |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
Value |
3 |
0 |
2 |
3 |
3 |
2 |
3 |
0 |
1 |
3 |
Dry and rough |
x |
|
x |
x |
x |
x |
x |
|
|
x |
Time of observation: approx.. 48 hours removal of the patches (day 25)
Challenge treatment – individual values (day 25)
Control animal No.: |
1 |
2 |
3 |
4 |
5 |
|
|
|
|
|
Value |
0 |
0 |
0 |
0 |
0 |
|
|
|
|
|
Treated animal No.: |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
Value |
3 |
0 |
2 |
3 |
3 |
3 |
3 |
1 |
2 |
3 |
Dry and rough |
x |
|
x |
x |
x |
x |
x |
|
X |
x |
Indurated |
x |
|
|
x |
|
|
x |
|
|
x |
After the challenge treatment nine animals of the treatment group (90%) showed a positive reaction during the observation period.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the conditions of the present study, none of ten animals of the treatment group showed a positive skin response after the challenge procedure.
Based on the results of this study Acid Blue 221 showed no evidence for skin sensitizing properties according to the classification criteria of Directive 2001/59/EC. - Executive summary:
Skin sensitization of Acid Blue 221 was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN.
Intradermal induction was performed using 5 % Acid Blue 221 in deionized water. Dermal induction and challenge treatment were carried out with 25 % Acid Blue 221 in deionized water.
The validity of the test system is confirmed by the periodically conducted positive control test using Alpha-hexylcinnamaldehyde for the maximization test.
Based on the results of this study, Acid Blue 221 showed no evidence for skin sensitizing properties according to the classification criteria of Directive 2001/59/EC.
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