Registration Dossier

Administrative data

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:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld
- Age at study initiation: 8 – 12 weeks
- Weight at study initiation: 19.1 g – 21.2 g
- Housing: single housed
- Diet (e.g. ad libitum): Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
40 % w/w preparation of the test substance in propylene glycol or with the vehicle alone
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: low water solubility
- Irritation: the results of a pretest with a 40% test-substance preparation in propylene glycol were considered, which did not show increased ear weights and lymph node weights as indication of ear irritation.
- Lymph node proliferation response: the results of a pretest with a 40% test-substance preparation in propylene glycol were considered, which did not show increased ear weights and lymph node weights.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: radioactive Murine Local Lymph Node Assay
- Criteria used to consider a positive response: The parameters used to characterize the response are lymph node cell count, 3H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. The increase SI of cell count by a factor of ≥ 1.5 and/or of 3H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.

TREATMENT PREPARATION AND ADMINISTRATION:
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
Concentration: As the 40 % test-substance preparation was the maximum technically applicable concentration, it was chosen for the main test, which was carried out as a limit test.
- Application volume: 25 μL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein.
- Terminal procedures: The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control induced positive responses in the auricular lymph nodes when applied as 3%, 10% or 30% preparations in acetone or methyl ethyl ketone.
lt is concluded that Alpha-Hexylcinnamaldehyde, techn. 85 % has a sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: control:1.00 test: 1.28 Calculation on basis of 4 animals due to irregularities during 3H-thymidine injection.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: control: 1052.1 test: 1343.7, Calculation on basis of 4 animals due to irregularities during 3H-thymidine injection.

Any other information on results incl. tables

Lymph node weight, Cell count, 3H-Thymidine incorporation and Ear weight: Stimulation indices (test group x / test group 1 (vehicle control))

Test

group

Treatment

 

Lymph node

Weight Index

Cell Count

Index

³H-thymidine inc. [DPM/lymph node pair] Index

Ear Weight
Index

1

2*

vehicle propylene glycol

40% in propylene glycol

1.00

1.05

1.00

1.23

1.00

1.28

1.00
0.99

* Calculation on basis of 4 animals due to irregularities during 3H-thymidine injection.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU