p-fluorophenyl 4-piperidyl ketone hydrochloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-12-01 (date test substance was received) to 2004-11-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: 1) No data on mitogenic stimulation or the metaphase-arresting substance, 2) Only 100 metaphase spreads scored per concentration.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

p-fluorophenyl 4-piperidyl ketone hydrochloride

EC Number:

247-070-0

EC Name:

p-fluorophenyl 4-piperidyl ketone hydrochloride

Cas Number:

25519-78-2

Molecular formula:

C12H14FNO.ClH

IUPAC Name:

4-(4-fluorobenzoyl)piperidine hydrochloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): T 1047 ((4-fluorophenyl)(4-piperidinyl) methanone hydrochloride)
- Physical state: white powder
- Analytical purity: approx. 100%
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark
- Other: received on 2003-12-01

Method

Species / strain

Species / strain / cell type:

other: human lymphocytes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % rat liver homogenate metabolising system (S9)

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 9.05, 19, 38, 76.15, 152.3, 304.6, 609.25, 1218.5 and 2437 μg/mL
The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test:
- Group 1 (4(20)-hour without S9): 0, 150, 300, 600, 1200, 1600 and 2000 µg/mL (1200, 1600 and 2000 µg/mL were selected for metaphase analysis.)
- Group 2 (4(20)-hour with S9): 0, 150, 300, 600, 1200, 1600 and 2000 µg/mL (1200, 1600 and 2000 µg/mL were selected for metaphase analysis.)
- Group 3 (24-hour without S9): 0, 37.5, 75, 150, 300, 450 and 600 µg/mL (150, 300 and 450 µg/mL were selected for metaphase analysis.)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: MEM culture medium
- Justification for choice of solvent/vehicle: The test substance was found to be soluble in culture medium and therefore MEM was selected as the vehicle for this study.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation at 10 μg/mL for the 4(20) hour exposure (Group 2)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; at 0.4 μg/mL for the 4(20) hour exposure and 0.2 μg/mL for the 24 hour continuous exposure (Groups 1 and 3 respectively)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in medium

DURATION:
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time: 20 hours was stated as the expression period in the study report. However, the study report failed to specify the time of addition of a spindle inhibitor.
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR: no data
STAIN: no data

NUMBER OF REPLICATIONS: Two cultures were tested per group. Except where there was the need to clarify an equivocal response, only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations.

NUMBER OF CELLS EVALUATED:
100 metaphases per analyzed culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (MI)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

No data was provided in the study report on the statistical tests performed. However, P<0.001 was considered statistically significant.

Results and discussion

Test results

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH at any of the dose levels used up to and including the 2437 µg/mL dose level.
- Effects of osmolality: There was no significant change in osmolarity at any of the dose levels used up to and including the 2437 µg/mL dose level.
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: the molecular weight of the test substance was 243.71 and therefore the maximum recommended dose level was 2437 µg/mL, which was equivalent to 10 mM.


RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity text was performed at dose levels of 9.5-2437 µg/mL using the same test design described above for the chromosome aberration assy. The mitotic index (MI) data showed evidence of dose-related test substance-induced toxicity in all three of the exposure groups. The dose response in Groups 1 and 2 was shown to be very steep. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 1218.5 µg/mL in Groups 1 and 2. In Group 3 the test substance was more toxic and metaphase cells were not observed at dose levels greater than 304.6 µg/mL. Therefore, the selection of the dose range for the chromosome aberration test was limited by toxicity for all three of the exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test mated itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance was seen to induce 54% mitotic inhibition at 2000 µg/mL in Group 1, 34% mitotic inhibition at 2000 µg/mL in Group 2 and 39% mitotic inhibition at 450 µg/mL in Group 3. A low MI value was seen for the Group 2 duplicate culture, which was considered to result in a slight underestimate of toxicity. Conversely, in Group 3, a high MI value for the duplicate culture of the 450 µg/mL dose group was also considered to result in a slight underestimate of toxicity. Therefore, it was considered that acceptable levels of toxicity were achieved in all cases. In all exposure groups the maximum dose level evaluated for chromosome aberrations was selected on the basis of toxicity. The test substance did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for induction of chromosome aberrations in human lymphocytes. The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolizing system after a 4(20)-hour exposure, or after a 24-hour exposure in the absence of metabolic activation. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.