1-[bis(4-fluorophenyl)methyl]piperazine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-10-18 to 2006-10-24

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The relative humidity in the animal room was between approximately 30-80%; however, this deviation did not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from the Federal Republic of Germany GLP Monitoring Authorities.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-[bis(4-fluorophenyl)methyl]piperazine

EC Number:

248-476-0

EC Name:

1-[bis(4-fluorophenyl)methyl]piperazine

Cas Number:

27469-60-9

Molecular formula:

C17H18F2N2

IUPAC Name:

1-[bis(4-fluorophenyl)methyl]piperazine

Test material form:

solid: particulate/powder

Details on test material:

White to light yellow powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00405251 ZT000841PUA421
- Expiration date of the lot/batch: 2007-06-30

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not indicated by the sponsor


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (Dimethylformamide) was quantitatively added. The test item concentrations were prepared serially. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made freshly before each dosing occasion.
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice (pretest excluded from Statement of Compliance). The data showed that the highest test item concentration, which can be used is 33 %. At the concentration of 33 % the treated mouse did not show any signs of irritation.

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: 7-8 weeks (beginning of acclimatization)
- Weight at study initiation: 19.6 +/- 1.1 grams
- Housing: single caging; cage type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen), bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +- 3 deg C
- Humidity (%): 30-80%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours artificial light

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1, 20, and 33 % (w/v)

No. of animals per dose:

4 females (nulliparous and non-pregnant) per group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 0.34 g/L in water, >500 g/L in methanol, >500 g/L in dichloromethane, >500 g/L in acetone, >500 g/L in ethanol, >500 g/L in 2-propanol, >500 g/L in N,N-dimethylformamide.
- Irritation: Pretest was performed in two mice, test item concentrations of 5, 10, 20, and 33% (w/v) were tested on one ear each. The treated mouse did not show any signs of irritation at 33% concentration.
- Lymph node proliferation response: no data


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Test item is regarded as a sensitizer in the LLNA if: First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR 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.


TREATMENT PREPARATION AND ADMINISTRATION:

Test Substance Preparation:
The test substance was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMF) was quantitatively added. The test substance concentrations were prepared serially. Homogeneity of the test substance in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.

Topical Application:
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, 20, and 33% (w/v) in DMF. 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 equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from Amersham International. Five days after the first topical application, all mice were administered with 250 uL of 78.9 uCi/mL 3HTdR (corresponds to 19.7 uCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3 HTdR:
Approximately five hours after treatment with 3HTdR, all mice were euthanized by intraperitoneal injection of Na-thiopental. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately + 4 degrees C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of "Ultima Gold" scintillation liquid and thoroughly mixed.

The level of 3HTdR incorporation was then measured on a B-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1-mL-aliquots of 5% trichloroacetic acid. The B-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

other: 2,4-dinitrobenzenesulfonic acid, sodium salt

Statistics:

The mean values and standard deviations were calculated for the body weight.

Results and discussion

Positive control results:

@-Hexylcinnamaldehyde
S.I. Results:
5% (w/v)= 2.04
10% (w/v) = 2.88
25% (w/v) = 3.56
EC3 value = 12.6% (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

4.6

Test group / Remarks:

1 %(w/v) in Dimethylformamide.

Results 2

Parameter:

SI

Value:

49.5

Test group / Remarks:

20 %(w/v) in Dimethylformamide.

Results 3

Parameter:

SI

Value:

43.9

Test group / Remarks:

33 %(w/v) in Dimethylformamide.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
DPM per lymph node
1% = 2034.8
20% = 22069.2
33% = 19561.9

STIMULATION INDEX:
1% = 4.6
20% = 49.5
33% = 43.9

EC3 CALCULATION: The EC3 Value could not be calculated, since all SI´s are above 3.

CLINICAL OBSERVATIONS: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

MORTALITY: No deaths occurred during the study period.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of *3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 4.6, 49.5, and 43.9 were determined with the test item at concentrations of 1, 20, and 33 % (w/v) in Dimethylformamide. The EC3 value could not be calculated, since all obtained SI´s were above 3. However, since the EC3 value is <1%, the test substance is classified as Skin sensitizer Cat. 1A.