Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 Jan to 10 Feb 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

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

Constituent 1
Chemical structure
Reference substance name:
(3R,4R)-1-benzyl-N,4-dimethylpiperidin-3-amine dihydrochloride
EC Number:
700-696-2
Cas Number:
1062580-52-2
Molecular formula:
C14H22N2.2HCl
IUPAC Name:
(3R,4R)-1-benzyl-N,4-dimethylpiperidin-3-amine dihydrochloride
Details on test material:
Batch No.: SKN045-008
Purity: Not specified

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Bicester, Oxon, UK.
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23 g
- Housing: Individual housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to mains food.
- Water (e.g. ad libitum): Free access to mains tap water.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): Approx. 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.

IN-LIFE DATES: From: 19 Jan 2009 To: 10 Feb 2009

Study design: in vivo (LLNA)

Vehicle:
other: 1% pluronic L92 in distilled water
Concentration:
Preliminary screening test: 50 % w/w
Main test: 50%, 25% , 10% w/w and 0% (vehicle)
No. of animals per dose:
Preliminary screening test: one mouse
Main test: four mice
Details on study design:
Preliminary screening test
The mouse was treated by daily application of 25 μL of the test material at a concentration of 50% w/w in 1% pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

Main test
Test material administration:
Groups of four mice were treated with the test material at concentrations of 50%, 25% or 10% w/w in 1% pluronic L92 in distilled water. The mice were treated by daily application of 25 μL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine administration:
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-Methyl Thymidine giving a total of 20 μCi to each mouse.

Terminal procedures:
Termination: Five hours following the administration of 3HTDR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of single cell suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTDR incorporation: After approximately 18 hours incubation at approx. 4℃, the precipitates were recovered by centrifugation at 2100 rpm for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTDR incorporation was measured by β-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in sample changer of the scintillator and left for approx. 20 minutes. The purpose of this period of time in darkness was to reduce the risk of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.
Positive control substance(s):
not specified
Statistics:
None stated

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI value for the vehicle control group was NA. The SI value for the experimental group treated with test substance concentrations 10% was 0.68. The SI value for the experimental group treated with test substance concentrations 25% was 1.05. The SI value for the experimental group treated with test substance concentrations 50% was 0.70.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The mean DPM/animal value for the vehicle control group was 5770.57 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 10% was 3916.51 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 25% was 6034.62 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 4029.25 DPM.

Any other information on results incl. tables

Clinical observations and Mortality data:

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Off white material on the ears was noted in animals treated with the test material at concentrations of 50% or 25% w/w in 1% pluronic L92 in distilled water.

Bodyweight:

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Disintegrations per minute, Disintegrations per minute/Node and Stimulation index:

Concentration (% w/w)

DPM

DPM/Node

SI

Result

0 (vehicle)

5770.57

721.32

NA

Negative

10%

3916.51

489.56

0.68

Negative

25%

6034.62

754.33

1.05

Negative

50%

4029.25

503.66

0.70

Negative

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance was considered to be a non-sensitiser under the conditions of the test.