Mixture of hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-5-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate and hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-7-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 5 to September 1, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to current OECD test guidelines and GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): E-BK105
- Physical state: Black powder, solid
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: July 6, 2015
- Storage condition of test material: Room temperature, shielded from light, tight container

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JNCrlj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.0 to 23.3 g
- Housing: Polycarbonate cages (92Wx205Dx127H mm) with after radio-isotope administration (220Wx325Dx130H mm) stainless steel mesh flooring, (stainless) steel racks, one animal per cage and after administration of radioisotope 6 animals per cage.
- Diet (ad libitum): Pellet diet (MF, Oriental Yeast Co., Ltd.), analysed: contaminants were confirmed to be within acceptable limits of the testing facility.
- Water (ad libitum): Tap water filtered through a 5 microm filter and disinfected by UV irradiation, analysed twice a year: contaminants were confirmed to be within acceptable limits of the testing facility.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6-23.8
- Humidity (%): 49.0-69.9
- Air changes (per hr): 6 to 30
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

=AOO

Concentration:

0, 5, 15, 50 w/v%.

No. of animals per dose:

6

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The dosing suspension was stirred with a magnetic stirrer throughout the application procedure.
- Irritation: 25 µl of the maximum feasible dose, 50 w/v%, was applied to two mice once a day for two days. This revealed no abnormalities in either animal, hence the maximum concentration was set at 50 w/v%.
- Lymph node proliferation response: no data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Stimulation Index (SI) value of 3 or greater was judged to be positive.

TREATMENT PREPARATION AND ADMINISTRATION:
Each animal was applied 25 µl to one side of both auricular using a micropipette. This was performed once daily for three consecutive days. The radioisotope was admnistered (250 µl/body) by injection five days after the initial auricular application.

Positive control substance(s):

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

Statistics:

Numerical data in the test substance groups were analysed by a multiple comparison test. Homogeneity of the variance among the groups was first tested by Bartlett's test. When variance was homogenous, which was, the one way analysis of variance was applied. The numerical data in the positive control group against the vehicle group was examined for homogeneity of the variance by F test and Welch test.

Results and discussion

Positive control results:

HCA group had a radioactivity mean of 5724.7 dpm; SI value was 5.36.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: SI of 5, 15 and 50 w/v% group is 1.14, 0.80 and 0.75, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean radioactivity values of AOO, 5 w/v%, 15 w/v% and 50 w/v% groups is 1068.7 dpm, 1215.5 dpm, 852.3 dpm, and 800.7 dpm, repsectively.

Any other information on results incl. tables

No abnormal clinical signs or lymph node weight or body weight were observed in any of the animals throughout the study period.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

E-BK105 has no sensitising potential under the conditions of this LLNA study (OECD 429).

Executive summary:

The sensitisation potential of E-BK105 was assessed in 30 female mice using the LLNA study performed according to OECD 429. E-BK105 was suspended in a mixture of acetone/olive oil (4:1) to make three concentrations of test substance: 5, 15 and 50 w/v%. HCA 25 v/v% was used as positive control and the vehicle was used as negative control. The SI values were 1.14, 0.80 and 0.75 in the 5, 15 and 50 w/v% test substance groups, respectively. The SI value of HCA was 5.36 showing the validity of the study. No abnormalities in body weight, lymph node weight or clinical signs were observed in any of the animals. From the results, it is concluded that E-BK105 has no sensitisation potential under the conditions of this study.