Dodeca (lithium, sodium)-2-({4-[4-(4-{bis[alkyl-(sulfonatoalkoxy)-4-({-sulfonato-[(substituted-phenyl)diazenyl]phenyl} diazenyl)anilino]-triazin-yl}-6-[alkyl-(sulfonatoalkoxy)-4-(sulfonato--[(substituted-phenyl)diazenyl]phenyl)diazenyl)anilino]piperazin-yl)-triazin-ylamino]-alkyl-5-(sulfonatoalkoxy)phenyl}diazenyl)-5-[(sulfonatophenyl)diazenyl]benzensulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 05 - September 08, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to 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

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): E-BW102
- Physical state: Red-brown powder
- Storage condition of test material: Room temperature, shielded from light, tight container
- Stability under storage conditions: No data

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: Young adult animals (8 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were individually housed in labeled Polycarbonate cages (92x205x127 mm) containing sterilised bedding for laboratory animals (Beta-Chip, Charles River Laboratories Japan, Inc.). On Day 6, animals were group housed in labeled Polycarbonate cages (220x325x130 mm) equiped with stainless steel mesh flooring, containing sterilised bedding for laboratory animals (Beta-Chip, Charles River Laboratories Japan, Inc.)
- Diet (e.g. ad libitum): Free access to pellet diet for experimental animals (MF, Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was 5 days.
- Health inspection: All of the animals were observed once daily with respect to their health conditions during this period and were confirmed to be in good health.

Results of analysis for diet and bedding were obtained from the supplier, and the levels of contaminants such as pesticide residues were confirmed to meet the specifications of the standard operating procedure of the test facility. The tap water was analyzed by Mitsubishi Chemical Analytech Co., Ltd. twice a year. From the analytical data, it was confirmed that the quality of water met the specifications of the standard operating procedure of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 – 24.0
- Humidity (%): 45.3 - 63.8
- Air changes (per hr): 6 to 30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: September 01, 2010 to September 06, 2010

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 5, 15 and 50 %

No. of animals per dose:

6

Details on study design:

RANGE FINDING TESTS:
According to the result of a preliminary study conducted at the test facility, the following 3 concentrations were set. In the preliminary study, 25 µL of the maximum feasible dose of the test substance suspended in acetone/olive oil at 50% was applied to two mice on the right side of auricular once a day for two days by open administration. The auricular skin reaction was then observed on the following day, and the result revealed no abnormalities in either animals. Hence, three concentrations of 50 (highest dose), 15 and 5% were set in this study.

MAIN STUDY
- Criteria used to consider a positive response: Based on the SI values obtained, the ratio (SI value) between each group was calculated, as the value of the control group is 1. An SI value of 3 or greater was judged to be positive.

ANIMAL ASSIGNMENT
Three groups of six animals were treated with one test substance concentration per group. One group of six animals was treated with vehicle and another group of six animals was treated with the positive control substance.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance was suspended in acetone/olive oil to give concentrations of 5, 15 and 50%. The dosing samples were prepared under UV-cut fluorescent light on the day of administration.

Induction - Days 1, 2 and 3:
Each test sample was applied at 25 μL (total 50 µL) to one side of the auricular using a micropipette. This treatment was performed once daily for three consecutive days. The dosing suspension was stirred with a magnetic stirrer throughout the application procedure. The vehicle and positive control animals were treated in the same way as the experimental animals, except that the vehicle and/or positive control substance was administered instead of the test substance.

Excision of the nodes - Day 6:
The radioisotope (3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Inc.) was administered five days (Day 6) after the initial auricular application. The radioisotope was injected at 250 µL (radioactivity: 0.74 MBq) to the caudal vein at a rate of approximately 1 mL/10 seconds using a disposable syringe with a 27G needle. At five hours after radioisotope injection, animals were euthanized by exsanguination from the addominal aorta under anesthesia with intraperitoneal administration of pentobarbital sodium. Thereafter, auricular lymph nodes were excised and weighed using an electronic balance.

Tissue processing for radioactivity - Day 6:
The weighed lymph nodes were transferred onto a cell strainer set on a centrifuge tube, and were mashed using the back of the piston of disposable syringe. The cell strainer and the back of the piston of disposable syringe were thoroughly washed with PBS. The suspension was adjusted to approximately 15 mL with PBS and then it was centrifuged (1000 rpm for 10 minutes at 4ºC). After centrifugation, the supernatant was removed and adjusted to approximately 15 mL with PBS again. The suspension was centrifuged (1000 rpm for 10 minutes at 4ºC) and supernatant was removed in as much as possible. Furthermore, the suspension was adjusted to approximately 15 mL with 5 w/v% TCA and it was stored in a refrigerator (4ºC) for approximately 18 hours. After storage the suspension was centrifuged (1000 rpm for 10 minutes at 4ºC) and supernatant was removed. A 1 mL portion of 5 w/v% TCA was added to the pellet to give cell lysate. In addition, o.5 mL each of tissue solubilizing agent and 2-Propanol were added into the cell lysate and it was dissolved while shaking for approximately 1 day.

Radioactivity measurements - Day 7
After shaking for approximately 1 day, the cell lysate was added with 10 mL of Hionic-Fluor (5 mLxtwice), and it was all transferred to a glass liquid scintillation vial (Wheaton). Then the radioactivity was measured using a liquid scintillation analyzer (quenched by tSIE method). Measurement was performed 5 minutes/time per vial. The same scintillation cocktail used for the sample solutions alone was measured 10 minutes/time for the background value (cpm). The value after subtracting the background value from the measured value of each dosing sample was taken as the radioactivity in the dosing sample. Additional, the detection limit of the radioactivity was at twofold of the background value.

Observations:
Body weights: On Day 1 (pre-dose), Day 3 and Day 6 (before radioisotope injection).
Clinical signs: Once daily from Days 1-6. However the animals were observed twice daily during the auricular application period (before application and within 1 hour after application).

Positive control substance(s):

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

Statistics:

The statistical analysis was performed on radioactivity.
Numerical data in the test substance groups were analyzed by a multiple comparison test for statistical significance. In the analysis, homogeneity of the variance among the groups was firstly tested by the Bartlett's test. Then, the variance was homogeneous, the one-way analysis of variance was applied. As no significant differences were resulted among the test groups in the analysis, further statistical analysis was not performed.
As for numerical data of the positive control group against the vehicle group, it was examined for homogeneity of the vaiance by F test and Welch test. The significant level of 5% (two-tailed) was employed for the statistical analysis. Statistical analyses were performed using the SAS preclinical package.

Results and discussion

Positive control results:

The SI value in the positive control group was 6.03.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values of the test substance groups were 0.91 at 5%, 1.07 at 15% and 0.63 at 50%, and none of the concentrations indicated the positive SI value equal to or greater than 3. The SI value of the positive control group was 6.03.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The radioactivity of the vehicle control group was 118 to 353 dpm (mean value 265.2 dpm) and the test substance groups was 94 to 323 dpm (mean value 240.8 dpm) at 5%, 153 to 393 dpm (mean value 283.0 dpm) at 15% and 100 to 229 dpm (mean value 166.0 dpm) at 50%. The radioactivity of the positive control group was 937 to 2412 dpm (mean value 1598.7 dpm).

Any other information on results incl. tables

Tables and figures of the Pre-screen test and Main study have been included in the attached document "LLNA tables and figures".

Results Pre-screen test:

The result revealed no abnormalities in the animals.

Other results - main study:

No abnormal clinical signs were observed in any animals throughout the experimental period.

 

No abnormal lymph node weight was shown in animals of any group.

No abnormal body weight was shown in animals of any group. 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Assessment of Contact Hypersensitivity to E-BW102 in the Mouse (Local Lymph Node Assay, 6 females/dose) was conducted according to OECD 429 guidelines and GLP principles.

E-BW102 was considered not to be a skin sensitizer, as none of the concentrations indicated the positive SI value equal to or greater than 3.

Based on these results E-BW102 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.