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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-03-31 to 2010-04-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study report which meets basic scientific principles. 3 animals per group.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in Jul 2010
Deviations:
yes
Remarks:
3 animals per group
GLP compliance:
no
Remarks:
no more details
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Analytical purity: 100%
- Lot/batch No.: 10SC8156928-2-2

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: approximately 9 weeks
- Weight at study initiation: 19.87 - 25.18 g (range)
- Housing: animals were housed 3 per cage in suspended aluminium cages with stainless steel mesh front and floor, w176 x d302 x h130 mm (Yamato Scientific Co., Ltd., Tokyo, Japan). Cages were exchanged with washed, sterile cages every week.
- Diet: CRF-1 diet (Oriental Yeasy Co., Ltd., Japan), ad libitum
- Water: filtered tap water via an automatic water supply equipment (Labo Engineering Inc.), ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 40 - 70
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
1, 5, 25% (w/v)
No. of animals per dose:
3 females
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the highest concentration that could be technically used was a 25% suspension in DMSO.
- Irritation: Three mice (1 per dose group) were treated by epidermal application to the dorsal surface of each ear with 1, 5 and 25% concentrations of the test item once daily for 3 consecutive days from Day 0. The ears were assessed for skin irritation on Day 1, 2 and 5. On Day 5, the draining auricular lymph nodes from each ear were excised and weighed. No signs of local irritation was observed in the animals. No treatment-related effects on body weight were noted. The doses applied were selected as the doses to be used in the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation.

- Criteria used to consider a positive response:
The test substance was considered to be positive for skin sensitisation when the SI ≥ 3.

TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of a 1, 5 and 25% concentration of the test substance in DMSO was applied to the dorsal surface of each ear of each mouse, for 3 consecutive days from Day 0. The control group was treated according to the same protocol with the vehicle alone. Local irritation reactions were assessed on Day 1 and 2. In addition, the body weight was measured on Day 1 and 5. On Day 5, 250 μL phosphate buffered saline (PBS) containing 80 μCi/mL of 3H-methyl thymidine (3H-TdR) was injected into the tail vein of each control and treatment mouse. Five hours later, the animals were sacrificed and the draining auricular lymph node of each ear was excised and pooled.The level of 3H-TdR-incorporation was measured in a β-scintillation counter.
The body weight was determined on Day 0 and 5, prior to the treatment.
Positive control substance(s):
not specified

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.5
Test group / Remarks:
1%
Parameter:
SI
Value:
3.8
Test group / Remarks:
5%
Parameter:
SI
Value:
4.1
Test group / Remarks:
25%
Cellular proliferation data / Observations:
Irritation was not observed and body weight gains were unaffected in all mice. Higher weights of auricular lymph nodes than vehicle control were observed and SI of 25% and 5% groups were higher than 3.

Any other information on results incl. tables

For the control, 1, 5, and 25% concentration groups, the DPM values per lymph node were 1554, 2314, 5887 and 6346, respectively. As the lymph nodes were pooled, these results were determined by dividing the measured value by the number of lymph nodes pooled.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The simulation index (SI) for the control, 1, 5, and 25% concentration groups was 1.00, 1.5, 3.8 and 4.1, respectively. As the threshold value is 3, the test substance is considered to be a skin sensitiser. The estimated concentration expected to produce a simulation index of 3 (EC3 value) is 3%.
Executive summary:

Skin sensitization potential of test item was evaluated in mice using the Local Lymph Node Assay (LLNA) using method similar to OECD429. CBA/JNCrljmice (three females/group) received the test substance solution (2 5μL/ear)in DMSO at 25%, 5% or 1% on the dorsal surface of each ear.The highest SI (Stimulation Index) of 4.1 was calculated for 25% group and it was concluded that test item is a skin sensitizer under the conditions of this study and the test item was considered to have an EC3 value of 3%.