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

Endpoint summary

Description of key information

The test substance is considered not to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

Specific details on test material used for the study:

No further details specified in the study report.

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

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.

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

Positive control results:

After the challenge treatment nine animals of the treatment group (90%) showed a positive reaction during the observation period.

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

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. 

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.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 Aug 2002 to 21 Aug 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Draft New Guideline 429: Skin Sensitization: Local Lymph Node Assay; Draft Document (November 2000).

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization" adopted by the Council on July 17, 1992 (reported Paris, April 29, 1993).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognized as the recommended test system.
Source: Harlan Netherlands, B.V. Postbus 6174, NL- 5960 AD Horst, The Netherlands
Number of animals for the pre-test (non-GLP): 2 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) group: 1
Age: 7 - 12 weeks (beginning of acclimatization)
Body weight: 13.2 g - 21.8 g (beginning of acclimatization period)
Identification: By unique cage card and individual color code.
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Room no.: E21 / RCC ltingen
Conditions: Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 ± 3 °C, relative humidity 30 - 70 % and 10 - 15 air changes per hour.
Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning.
These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was a 12 hour fluorescent light I 12 hour dark cycle with at least 8 hours music during the light period.
Accommodation: In groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
Diet: Pelleted standard Kliba 3433, batch no. 34/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. Results of analyses for contaminants are archived at RCC.
Water: Community tap water from Itingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.

Vehicle:

other: ethanol:water, 7:3 (v/v)

Concentration:

To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in 2 mice with concentrations of 1 %, 5 %, 10% and 25% (w/v) (pretest excluded from Statement of Compliance).
The test item in the main study was assayed at three consecutive concentrations (5%, 10%, 25%). The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

No. of animals per dose:

Four animals per dose

Details on study design:

TEST ITEM PREPARATION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (ethanol:water, 7:3 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made shortly before each dosing.

STUDY CONDUCT
TREATMENT PROCEDURES
TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %, 10 % and 25 % (w/v) in ethanol:water, 7:3 (v/v). The application volume, 25 μI, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 31 0; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 μI of 73.51 μCi/ml 3HTdR (equal to 18.4 μCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, ZOrich). The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).
Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing three times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C overnight for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OBSERVATIONS
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Twice daily from acclimatization start to the termination of in-life phase.
Body weights: At acclimatization start and prior to necropsy.
Clinical signs (local I systemic): Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.

INTERPRETATION OF RAW DATA
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPMINODE) and as the ratio of 3HTdR into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPMINODE values were determined, mean scintillation background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
The decision to select a stimulation index of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 =(a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.l. value of 3 on the local lymph node assay dose response plot.

Positive control results:

ALPHA-HEXYLCINNAMALDEHYDE was found to be a non-sensitizer when tested up to 10% (w/v) in acetone:olive oil, 4:1 (v/v).
ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at concentration of 25% (w/v).
EC3 is the estimated concentration for a STIMULATION INDEX of 3. In this study EC3 of 11.3% (w/v) was theoretically calculated with STIMULATION INDICES of 2.6 and 7.1 at test item concentrations of 10 % and 25 % (w/v).

Key result

Parameter:

EC3

Value:

8.75

Test group / Remarks:

Test Item Concentrations of 5% and 10% (w/v). The S.I. values obtained at test item concentrations of 5% and 10% were used to calculate the EC3 value.

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

Test Item Concnetration 5% (w/v)

Key result

Parameter:

SI

Value:

3.2

Test group / Remarks:

Test Item Concentration 10% (w/v)

Key result

Parameter:

SI

Value:

3.3

Test group / Remarks:

Test Item Concentration 25% (w/v)

Cellular proliferation data / Observations:

VIABILITY/MORTALITY
No deaths occurred during the study period.

CLINICAL SIGNS
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

CALCULATION AND RESULTS OF INDIVIDUAL DATA

The following results were obtained:

Vehicle: ethanol:water, 7:3 (v/v)

 

Test item concentration % (w/v)		Measurement dpm^a)	Calculation			Result
			dpm – BG	Number of lymph nodes	dpm per lymph node^b)	S.I.
--	BG I	12	--	--	--	--
--	BG II	10	--	--	--	--
--	CG 1	1127	1116	8	140	--
5	TG 2	2651	2640	8	330	2.4
10	TG 3	3563	3552	8	444	3.2
25	TG 4	3652	3641	8	455	3.3

BG = Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

^a)= The mean value was taken from the figures BG I and BG II

^b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/Node was determined by dividing the measured value by the number of lymph nodes pooled.

 

	Test item concentration % (w/v)	S.I.
Group 2	5 (a)	2.4 (b)
Group 3	10 (c)	3.2 (d)
EC 3 = (a-c) [(3-d)/(b-d)] + c = 8.75 % (w/v)

EC3 = Estimated concentration for a STIMULATION INDEX of 3.

a,b,c,d = Co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

 

BODY WEIGHTS (GRAM)

FEMALES

	ACCLIMATISATION	TREATMENT
DAYS WEEKS ANIMAL	1 1	6 1
GROUP 1 (CONTROL GROUP)
1 2 3 4	21.5 16.7 19.7 17.6	22.5 17.6 22.1 19.8
GROUP 2 (TEST GROUP 5%)
5 6 7 8	15.3 16.1 13.2 19.0	16.9 17.7 14.8 21.0
GROUP 3 (TEST GROUP 10%)
9 10 11 12	19.1 18.7 17.8 17.6	20.3 21.5 19.8 19.2
GROUP 4 (TEST GROUP 25%)
13 14 15 16	17.9 21.8 16.6 18.6	19.4 21.0 18.6 20.7

 

BODY WEIGHTS (GRAM) SUMMARY

FEMALES

ACCLIMATISATION		GROUP 1 CONTROL GROUP	GROUP 2 TEST GROUP 5%	GROUP 3 TEST GROUP 10%	GROUP 4 TEST GROUP 25%
DAY 1 WEEK 1	MEAN ST. DEV. N	18.8 2.1 4	15.9 2.4 4	18.3 0.7 4	18.7 2.2 4

 

TREATMENT		GROUP 1 CONTROL GROUP	GROUP 2 TEST GROUP 5%	GROUP 3 TEST GROUP 10%	GROUP 4 TEST GROUP 25%
DAY 6 WEEK 1	MEAN ST. DEV. N	20.5 2.3 4	17.6 2.5 4	20.2 1.0 4	19.9 1.1 4

 

Positive Control Study

CALCULATION AND RESULTS OF INDIVIDUAL DATA

The following results were obtained:

Vehicle: acetone:olive oil, 4:1 (v/v)

 

Test item concentration % (w/v)		Measurement dpm^a)	Calculation			Result
			dpm – BG	Number of lymph nodes	dpm per lymph node^b)	S.I.
--	BG I	3	--	--	--	--
--	BG II	3	--	--	--	--
--	CG 1	4232	4229	8	529	--
5	TG 2	12169	12166	8	1521	2.9
10	TG 3	10977	10974	8	1372	2.6
25	TG 4	29856	29853	8	3732	7.1

BG = Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

^a)= The mean value was taken from the figures BG I and BG II

^b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/Node was determined by dividing the measured value by the number of lymph nodes pooled.

 

	Test item concentration % (w/v)	S.I.
Group 2	5	2.9
Group 3	10 (a)	2.6 (b)
Group 4	25 (c)	7.1 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 11.3 % (w/v)

EC3 = Estimated concentration for a STIMULATION INDEX of 3.

a,b,c,d = Co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study STIMULATION INDICES OF 2.4, 3.2 and 3.3 were determined with the test item at concentrations of 5%, 10% and 25% (w/v) in ethanol:water, 7:3 (v/v).
A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
Based on these criteria, the test item Supranol Blau GLW was found to be a non-sensitizer when tested up to 5% (w/v) in ethanol:water, 7:3 (v/v).
Blau GLW showed an allergenic potency when tested at concentrations of 10% and 25% (w/v).
EC3 is the estimated concentration for a STIMULATION INDEX of 3. In this study EC3 of 8.75% (w/v) was theoretically calculated with STIMULATION INDICES of 2.4 and 3.2 at test item concentrations of 5% and 10% (w/v).

Executive summary:

In order to study a possible allergenic potential of Supranol Blau GLW three groups of four female mice each were treated with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 7:3 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter.

 

No test item-related clinical signs were observed.

 

All treated animals survived the scheduled study period.

 

A test item is regarded as a sensitizer if the exposure to at least one concentration resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

 

	Test Item Concentration % (w/v)	S.I.
Group 2	5*	2.4*
Group 3	10*	3.2*
Group 4	25	3.3
EC3 = 8.75 % (w/v)
A clear dose-response relation was observed. * The value was used in calculation of EC3.

 

 

CONCLUSION

In this study STIMULATION INDICES of 2.4, 3.2 and 3.3 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol:water, 7:3 (v/v).

 

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.

 

Based on these criteria, the test item Supranol Blau GLW was found to be a non-sensitizer when tested up to 5 % (w/v) in ethanol:water, 7:3 (v/v).

Supranol Blau GLW showed an allergenic potency when tested at concentrations of 10% and 25% (w/v).

 

EC3 is the estimated concentration for a STIMULATION INDEX of 3. In this study EC3 of 8.75% (w/v) was theoretically calculated with STIMULATION INDICES of 2.4 and 3.2 at test item concentrations of 5 % and 10 % (w/v).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitization potential of Acid Blue 221 was investigated 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.

RESULTS

DETERMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS

Intradermal injections with the 5.0 % and 1 % preparation caused clear edema, the 0.2 % preparation caused slight edema. Due to the color of the test substance the treated skin of the animals could not be assessed for erythema. Based on this preliminary test, a 5 % preparation was selected for the intradermal injections in the main test.

 

DETERMINATION OF THE PRIMARY NON-IRRITATING CONCENTRATION

No signs of irritation occurred after administration of the different test concentrations.

Based on these results, a concentration of 25 % Acid Blue 221 in deionized water was chosen for the challenge at day 22.

 

MAIN TEST FOR THE SENSITIZING PROPERTIES

Body weight gains and clinical signs

The body weight gains of the animals were not impaired.

The treated animals showed no clinical signs of intoxication throughout the study.

 

Intradermal induction treatment

Intradermal injections with Freund's Adjuvant (without test substance) caused severe erythema and edema as well as indurations and encrustations. Intradermal injections of the vehicle alone exhibited no signs of irritation.

Intradermal injections with Freund's Adjuvant (with test substance) caused severe edema as well as indurations and encrustations. The administration sites treated with the test substance in deionized water showed clear edema as well as indurations and encrustations.

Due to the color of the test substance the treated skin of the animals could not be assessed for erythema.

Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at day 7.

 

Dermal induction treatment

After the removal of the patches at day 10, severe erythema and edema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with Freund's Adjuvant. The administration sites treated with the test substance in deionized water showed clear edema, indurated and encrusted and necrosis. Intradermal injections of the sesame oil alone exhibited no signs of irritation.

Due to the color of the test substance the treated skin of the animals could not be assessed for erythema.

 

Dermal challenge treatment

No skin reactions were observed in the control and the treatment group 24 and 48 hours after removal of the occlusive bandage.

 

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.

In order to study a possible allergenic potential of Acid Blue 221, three groups of four female mice each were treated with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 7:3 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter.

 

No test item-related clinical signs were observed.

 

All treated animals survived the scheduled study period.

 

A test item is regarded as a sensitizer if the exposure to at least one concentration resulted in an incorporation of³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

 

	Test Item Concentration % (w/v)	S.I.
Group 2	5*	2.4*
Group 3	10*	3.2*
Group 4	25	3.3
EC3 = 8.75 % (w/v)
A clear dose-response relation was observed. * The value was used in calculation of EC3.

 

 

CONCLUSION

In this study STIMULATION INDICES of 2.4, 3.2 and 3.3 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol:water, 7:3 (v/v).

 

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.

 

Based on these criteria, the test item was found to be a non-sensitizer when tested up to 5 % (w/v) in ethanol:water, 7:3 (v/v).

SupranolBlau GLW showed an allergenic potency when tested at concentrations of 10% and 25% (w/v).

 

EC3 is the estimated concentration for a STIMULATION INDEX of 3. In this study EC3 of 8.75% (w/v) was theoretically calculated with STIMULATION INDICES of 2.4 and 3.2 at test item concentrations of 5 % and 10 % (w/v).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the Guinea Pig Maximisation Test (GPMT), tested with 5% test substance to intradermal induction and 25% test substance for dermal induction and challenge, no animal showed a positive reaction. In the Local Lymph Node Assay (LLNA) in mice, tested at 5, 10, and 25% test substance concentrations, a stimulation index of 2.4, 3.2, and 3.3 were, respectively, were observed, indicating a weak positive reactions at test substance concentrations above the EC3 value of 8.75%.

However, as discussed by Kreiling et al., 2008 and Basketter et al. 2009, there are false positive results in the LLNA, especially in surfactants with unsaturated C=C double bounds.

 

Based on the fact that there was absolutely no effect seen in the GPMT, this substance is considered not to be a skin sensitiser.