Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA:J
Sex:
female
Vehicle:
dimethyl sulphoxide
Concentration:
Eugenol(positive control): 25%
Test substance: 2.5, 5, 10%(w/v)
No. of animals per dose:
Acetone: Olive oil(4:1, v/v): 4
Eugenol(25%(w/v)): 4
Test substance(2.5, 5, 10%): each 4
DMSO: 4
Details on study design:
1. Method
1.1. Administration of Substance: Day 1, Day 2, Day 3
The test substance (the vehicle control and the positive control) was spread over the entire dorsal surface of the ear using a micropipette at 25 uL/ear.
1.2. No Treatment: Day 4
1.3. BrdU Solution Administration: Day 5
Injection 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution inter-peritoneally.
1.4. Auricular Lymph Node Extraction: Day 6
Approximately 24 hours after BrdU injection administering euthanasia to the animals. Excising the draining auricular lymph nodes from each mouse ear.
1.5. Preparation of Cell Suspension : Day 6
For each mouse, a single-cell suspension of lymph node cells(LNC) excised bilaterally was prepared by 70μm nylon mesh to generate a single cell suspension. In each case, the target volume of the LNC suspension was adjusted to the determined optimized volume. The optimized volume (15 mL) was based on the mean absorbance within 0.1–0.2 in the NC group.
1.6. Cellular Proliferation Measurement: Day 6
BrdU was measured by ELISA using a commercial kit. Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. And dispensing, centrifugation (300 g, 10 min) would be allowed the cells to settle to the bottom and remove the PBS. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and then the substrate solution was added and allowed to produce chromogen. Absorbance at 370 nm (Emission wavelength, em) with a reference wavelength of 492 nm (Reference wavelength, ref) was measured.
2. Observation
2.1. Clinical Signs
Clinical signs were daily observed on clinical signs.
2.2. Body Weight
Body weights were recorded on Day 1, immediately prior to dosing and on Day 6.
2.3. Observation of Test Substance Application site
Skin responses to site of test substance application were observed once each day according to “Erythema scores”.
Positive control substance(s):
eugenol (CAS No 97-53-0)
other: Acetone : Olive Oil(4:1, v/v)
Positive control results:
1. Mortality: No deaths were observed in Acetone
2. Clinical signs: No clinical signs were observed in Acetone.
3. Body weight: No significant change was observed.
4, Ear
Key result
Parameter:
SI
Value:
0.998
Test group / Remarks:
2.5%(w/v)
Key result
Parameter:
SI
Value:
1.073
Test group / Remarks:
5%(w/v)
Key result
Parameter:
SI
Value:
1.441
Test group / Remarks:
10%(w/v)
Key result
Parameter:
SI
Value:
2.083
Test group / Remarks:
Positive control(Eugenol, 25(w/v))
Interpretation of results:
GHS criteria not met
Conclusions:
It was evaluated that were negative for skin sensitization under this test condition.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification