Tetra(sodium/potassium)-7-[(E)-{2-acetamido-4-[(E)-(4-{[4-chlor-6-({2-[(4-fluor-6-{[4-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonato-1-naphthyl)diazenyl]-5-methoxyphenyl}diazenyl]-1,3,6-naphthalintrisulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 10 May 2006; Experiment completion date - 24 May 2006; Study completion date - 15 June 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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description: Black sticky powder
Purity: Content of organic part (Na-salt): approx. 80 %;Oligomers: 13 %;Main component: approx. 53 %
Stability: Stable under storage conditions.
Storage: At room temperature, desiccated and light protected.
Description: Black sticky powder
Purity: Content of organic part (Na-salt): approx. 80 %;Oligomers: 13 %;Main component: approx. 53 %
Stability: Stable under storage conditions.
Storage: At room temperature, desiccated and light protected.

Specific details on test material used for the study:

Identity: FAT 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

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: 3 females;
Number of animals for the main study: 16 females;
Number of animals per group: 4 females (nulliparous and non-pregnant);
Age: 8-12 weeks (beginning of acclimatization);
Body weight: 16-24 g;
Identification: Each cage by unique cage card;
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: Under laboratory conditions after health examination. Only healthy animals were used for the study.

Conditions
Standard Laboratory Conditions.
Air-conditioned with ranges for room temperature of 22 ± 3 °C and relative humidity 30-70 % and approximately 10-15 air changes per hour. There was a 12 hours artificial fluorescent light/12 hours dark, music during the light period.
Accommodation: Individual in Makrolon type-2 cages with standard softwood bedding.
Diet: Pelleted standard Provimi Kliba 3433 mouse maintenance diet (batch 001/06) ad libitum.
Water: Community tap water, ad libitum.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

2.5 %, 5 % and 10 % in 25 µl of DMF

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 concentration of 2.5 %, 5 % and 10 % in DMF by topical application to the dorsum of each ear lobe for three consecutive days. Five days after the first topical application, the mice were injected intravenously into a tail vein with radiolabelled 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:

S.I. of 1.8, 2.9 and 6.2 of determined with alpha-hexylcinnamaldehydewere at concentrations of 5 %, 10 % and 25 %, respectively, in acetone: olive oil, 4:1 (v/v). 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:

SI

Value:

10.9

Test group / Remarks:

2.5 %

Results 2

Parameter:

SI

Value:

16.8

Test group / Remarks:

5 %

Results 3

Parameter:

SI

Value:

23

Test group / Remarks:

10 %

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

S.I. of 10.9, 16.8 and 23.0 were determined with the test item at concentrations of 2.5 %, 5 % and 10 %, respectively, in DMF and the test item was therefore found to be a potential skin sensitizer in the LLNA tests.

Executive summary:

In a GLP compliant study conducted according to OECD test guideline 429, three groups each of four female mice were treated daily with the test item at concentration of 2.5 %, 5 % and 10 % in DMF by topical application to the dorsum of each ear lobe for three consecutive days. Five days after the first topical application, the mice were injected intravenously into a tail vein with radiolabelled 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. All treated animals survived the scheduled study period. No clinical signs were observed. After the first topical application, the dark red stain was found at both dosing sites in all mice of the test item group, persisting for the remainder of the in-life phase of the study. In the study, S.I. of 10.9, 16.8 and 23.0 were determined with the test item at concentrations of 2.5 %, 5 % and 10 %, respectively, in DMF. The EC3 value cannot be determined, since this calculation requires a S.I. values less than 3. Although according to the data reported above, the EC3 can be derived to be <1 %, thus, the substances shows a high sensitizing potential. Based on these results, the substance is to be classified as Skin Sensitizer category 1A according to the CLP regulation (Regulation EC No. 1272/2008 as amended).