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

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:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

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

Test material information

Constituent 1

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

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)

Induction

Induction 1

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

Induction 2

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

Induction 3

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)

Results

Results 1

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

Results 2

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

Results 3

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.

Results 4

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.

Results 5

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

Results 6

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.