N-(1-benzylpiperidin-4-yl)-N-phenylpropionamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-08-10 to 2005-8-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

Relative humidity 30-84%

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-(1-benzylpiperidin-4-yl)-N-phenylpropionamide

EC Number:

216-014-7

EC Name:

N-(1-benzylpiperidin-4-yl)-N-phenylpropionamide

Cas Number:

1474-02-8

Molecular formula:

C21H26N2O

IUPAC Name:

N-(1-benzylpiperidin-4-yl)-N-phenylpropanamide

Details on test material:

- Name of test material (as cited in study report): T424
- Substance type: No data
- Physical state: solid at room temperature
- Color: white, as determined by an RCC laboratory staff member
- Analytical purity: 100%
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: RT000424G1A251
- Expiration date of the lot/batch: 2005-12-31
- Stability under test conditions: No data
- Storage condition of test material: room temperature
- Solubility: 0.13 g/L in water; >500 g/L ethanol
- Other: No data

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT000424G1A251, Janssen Pharmaceutica N.V.
- Expiration date of the lot/batch: 31-December-2005
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: N/A
- Solubility of the test substance in the solvent/vehicle: 0.13g/L in water, >500 g/L in ethanol

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Homogeneity of the test item in the vehicle was maintained during treatment with the magentic stirrer.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL-5960 AD Horst, The Netherlands
- Age at study initiation: 5-7 weeks (begininning of acclimatisation)
- Weight at study initiation: 16.3-19.1 g
- Sex: females (nulliparous and non-pregnant)
- Number of animals for the main study: 16
- Housing: Single caging (Makrolon Type 1, with wire mesh top) (EHRET GmbH, D-79302 Emmendingen)
- Diet (e.g. ad libitum): pellet standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-64380 Rossendorf)
- Acclimation period: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3
- Humidity (%): 30-84
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2005-08-10 To: 2005-08-16

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10, 25, 33% (w/v) test substance in vehicle

No. of animals per dose:

4 animals

Details on study design:

RANGE FINDING TESTS: LLNA
- Compound solubility: 0.13 g/L in water; >500 g/L in ethanol
- Irritation: No irritation was observed.
- Lymph node proliferation response: No irritation effects were observed at these concentrations after a single application.
- Other: Only two mice were used to test 4.125, 8.25, 16.5 and 33% (w/v), tested on one ear each.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1. The exposure to at least one concentration of the test item resulted in a n incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
2. The data were compatible with a conventional dose response, although allowance must have been made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS
Mortality/Viability: Daily from acclimatisation start to the termination of in-life phase.
Body weights: Prior to the first application and prior to necropsy
Clinical signs: Daily from acclimatisation start to the termination of in-life phase. Especially the treatment sites were observed carefully.

TREATMENT PREPARATION AND ADMINISTRATION:
Each group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25 and 33% (w/v) in acetone:olive oil 4:1 (v/v). The application volume, 25 uL, was spread over the entire dorsal surface (diameter ~8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an quivalent volume of the relevant vehicle alonge. A hair dryer was used to dry the ear surface to avoid the loss of item applied.

3H-methyl thymidine (3HTdR) was purchased from Amersharm International (Amersham product code no. TRA 310; specific acitivity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were adiminstered with 250 ul of 81.3 uCi/ml 3HTdR (corresponds to 20.325 uCi 3HTdR per mouse) by intravenous injection via a tail vein.

Approximately five hours after treatment all mice were euthanised. The draining lymph nodes were rapidly excised and pooled for each experimental group. Single cell suspensions of pooled lymp node cells were prepared by gentle mechanical disaggregation through stailness steel gauze. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5% trichloroacetic acid and incubated at ~4 degrees C for at last 18 hours for precicipation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid and transferred to glass scintillation vials with 10 ml of Ultima Gold scintillation liquid and throughly mixed. The level of 3HTdR incorporation was meansured as well as background 3HTdR levels.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation:
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a,b) and (c,d) are respectively the co-ordinates of the two pair of data lying imediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

Not applicable

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.99

Test group / Remarks:

4 animals in 10% v/v in acetone/olive oil 4:1 group

Results 2

Parameter:

SI

Value:

0.95

Test group / Remarks:

4 animals in 25% v/v in acetone/olive oil 4:1 group

Results 3

Parameter:

SI

Value:

1.05

Test group / Remarks:

3 animals in 33% v/v acetone/olive oil 4:1 group

Results 4

Parameter:

other: DPM

Value:

987.5

Test group / Remarks:

4 animals 10% v/v acetone/olive oil 4:1 group

Results 5

Parameter:

other: DPM

Value:

944.2

Test group / Remarks:

4 animals in 25% v/v acetone/olive oil 4:1 group

Results 6

Parameter:

other: DPM

Value:

1 043.1

Test group / Remarks:

3 animals 33% v/v acentone/olive oil 4:1 group

Cellular proliferation data / Observations:

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no system findings were observed. Body weight of the animals was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

The EC3 value could not be calculated since all SI (Simulation Index) values were below 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item (T424) is not a skin sensitiser under the described conditions.