Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date - 15 Mar 2006;
Experiment completion date - 29 Mar 2006
Study completion date - 19 May 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Cas Number:

882878-51-5

Molecular formula:

C39H28CuF2N14Na4016S4

IUPAC Name:

Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description: Black powder
Stability of test item: Stable under storage conditions
Storage conditions: At room temperature (range of 20 ±5 °C), light and moisture protected. The test item is very hygroscopic! Stored in a desiccator.

Specific details on test material used for the study:

Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaHsdRcc(SPF)
Number of animals for the pre-test (non-GLP): 3 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of negative control group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Body weight: 16 g - 24 g (ordered)
Identification: Each cage by unique cage card.
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
Conditions: Standard Laboratory Conditions. Air-conditioned with ranges for room temperature 22 ±3 °C, relative humidity 30 - 70 % and 10 - 15 air changes per hour. There was a 12 hour fluorescent light /12 hour dark cycle with at least 8 hours music during the light period.
Accommodation: Individual in Makrolon type-2 cages with standard softwood bedding.
Diet: Pelleted standard Kliba 3433, batch no. 76/05 mouse maintenance diet available ad libitum.
Water: Community tap water from Itingen, available ad libitum.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0 (Control group), 5 %, 10 % and 25 %

No. of animals per dose:

4

Details on study design:

Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine, ³HTdR). 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 subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a ß-scintillation counter.

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.

Results and discussion

Positive control results:

In the study STIMULATION INDICES of 1.8, 2.9 and 6.2 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in acetone:olive oil, 4:1 (v/v). And Alpha-hexylcinnamaldehyde was therefore found to be a skin sensitizer in the LLNA tests and an EC3 value of 10.5 % was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks on result:

other: No dose-response relationship was observed. An EC3 value could not be determined because this calculation requires a S.I. value of less than 3.

Results 2

Parameter:

SI

Value:

6.2

Test group / Remarks:

Group 2 : 5 % test item concentration

Results 3

Parameter:

SI

Value:

10.1

Test group / Remarks:

Group 3 : 10 % test item concentration

Results 4

Parameter:

SI

Value:

8.1

Test group / Remarks:

Group 4 : 25 % test item concentration

Any other information on results incl. tables

VIABILITY / MORTALITY
No deaths occurred during the study period.

CLINICAL SIGNS
No clinical signs 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 prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

SIZE OF THE DRAINING LYMPH NODES
The size of the draining lymph nodes of Group 2 was 2-fold large compared to that of the control group. The size of the draining lymph nodes of Groups 3-4 was 3-fold large compared to that of the control group.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in DMF and the test item was therefore found to be a potential skin sensitizer in the LLNA tests.

Executive summary:

In order to study a possible contact allergenic potential of the test item, a LLNA study was conducted according to OECD test guideline 429 in a GLP-certified laboratory. Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. All treated animals survived the scheduled study period. No clinical / local signs were observed. After each topical application, the residual test item was found at all the local dosing sites in all mice of Group 3 (10 %) and Group 4 (25 %). After the second and the third topical application, the residual test item was found at all the local dosing sites in all mice of Group 2 (5 %). In the study, S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5%, 10% and 25%, respectively, in DMF. The test item was therefore found to be a potential skin sensitizer in the LLNA test and it shall be included in the Skin sensitisers Category 1 according to the CLP Regulation (Regulation EC No. 1272/2008)