1-benzyl-N-phenylpiperidin-4-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-03-15 to 2006-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-benzyl-N-phenylpiperidin-4-amine

EC Number:

214-583-6

EC Name:

1-benzyl-N-phenylpiperidin-4-amine

Cas Number:

1155-56-2

Molecular formula:

C18H22N2

IUPAC Name:

1-benzyl-N-phenylpiperidin-4-amine

Details on test material:

- Name of test material (as cited in study report): T000293; N-phenyl-1-(phenylmethyl)-4-piperidinamine
- Substance type: no data
- Physical state: solid
- Analytical purity: 100%
- Lot/batch No.:00479757 RT000293G1A401
- Expiration date of the lot/batch: 2006-06-30
- Stability under test conditions: no data
- Storage condition of test material: at room temperature (range of 20 +/- 5 deg C), light protected
- Other:
- Stability of test item: stable under storage conditions

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaHsdRcc (SPF)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/CaHsdRcc(SPF) mice from RCC Ltd. Laboratory Animal Service
- Age at start of acclimatization: 8-12 weeks
- Weight at study initiation (ordering): 16-24 g
- Housing: standard laboratory conditions, individual in Makrolon Type 2 cage, with standard soft wood bedding
- Diet: ad libitum, pelleted standard Kilba 3433 mouse maintenance diet
- Water: ad libitum, community tap water from Itingen
- Acclimation period: 2006-03-15 to 2006-03-21, under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22+/- 3 deg C
- Humidity (%): 30 -70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12hr fluorescent light/12 hr dark with at least 8 hours music during the light period.


IN-LIFE DATES: From: 2006-03-22 To: 2006-03-27

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1%, 3%, and 10%

No. of animals per dose:

pretest: 3 females
main study: 16 females (4 females per group)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: In a non-GLP solubility pre-test, the test substance was tested in different vehicles: acetone/olive oil (4/1,v/v), ethanol/water (7/3, v/v) and N,N-dimethylformamide (DMF). DMF was found to be a suitable vehicle and was selected and used in the main test. 50% was the highest technically achievable concentration in the chosen vehicle.
- Irritation: Three single animals were each treated with one of the three different concentrations: 10%, 25%, and 50% in DMF, in both ears on three consecutive days. After the second and the third topical application, slight to severe ear erythema was observed at all the dosing sites, respectively. 10% was the highest dosing concentration in the main tests for avoiding systemic toxicity and excessive local irritation.
- Lymph node proliferation response: no data


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: First, that exposure to at least one concentration of the test item resulted in an incorporation of HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. The decision to select a stimulation Index (S.I) of 3 as an arbitrary indication of sensitizing activity was made.


TREATMENT PREPARATION AND ADMINISTRATION:

The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle N,N-dimethylformamide (DMF) was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer.
The preparations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test substance in the vehicle was maintained until start of treatment using an appropriate homogenizer.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test substance concentrations of 1, 3 and 10% (w/v) in N,N-dimethylformamide (DMF). The application volume, 25 uL, was spread over the entire dorsal surface (Ø~8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Positive control substance(s):

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

Statistics:

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 coordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

DPM (corrected for background):
vehicle control: 4687
5% test group: 8487
10% test group: 13610
25% test group: 29209

SI
Test Group 2 (5% (w/v)): S.I.= 1.8
Test Group 3 (10% (w/v)): S.I. = 2.9
Test Group 4 (25% (w/v)): S.I. = 6.2

EC3 was estimated to be 10.5%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.5

Test group / Remarks:

4 animals in 1% w/v in DMF group

Results 2

Parameter:

SI

Value:

2

Test group / Remarks:

4 animals in 3% w/v in DMF group

Results 3

Parameter:

SI

Value:

3.3

Test group / Remarks:

4 animals in 10% w/v in DMF group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
-1% w/v = 3641
-3% w/v = 4689
-10% w/v = 7804
-Control = 2405

EC3 CALCULATION
-The EC3 Value = 8.4%

CLINICAL OBSERVATIONS:
-Viability / Mortality: No deaths occurred during the study period.
-Clinical Signs: No clinical signs of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
-Body Weights:The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Test item concentration % (w/v)	Group	Measurement DPM	DPM-BG a)	number of lymph nodes	DPM per lymph node b)	S. I.
---	BG I	33	---	---	---	---
---	BG II	38	---	---	---	---
---	CG 1	2405	2369	8	296
1	TG 2	3641	3605	8	451	1.5
3	TG 3	4689	4653	8	582	2.0
10	TG 4	7804	7768	8	971	3.3

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II.

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

The EC3 Value = 8.4%.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test substance was found to be a potential skin sensitizer in the LLNA tests and an EC3 value of 8.4% was derived. Based on CLP regulation, the test item is considered to be classified as a skin sensitizer category 1B.