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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-05-29 to 2006-06-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only 3 strains are tested, however an expert statement is added in 'Overall Remarks' to justify the fact that no further testing is necessary.

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

ninth addendum

Deviations:

yes

Remarks:

only three S. typhimurium (TA98, TA100, and TA 102) strains were used.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

only three S. typhimurium (TA98, TA100, and TA 102) strains were used

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

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 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, and TA 102

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment / Experiment I (with and without metabolic activation for all tester strains): 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate;
Experiment II (with and without metabolic activation for all tester strains): 0, 16, 50, 166, 500, 1660 and 5000 µg/plate;

Since the test item was soluble in DMSO up to the standard limit concentration recommended in the regulatory guidelines that this assay followed (5000 µg/plate), the highest tested concentration in the Pre-experiment/Experiment I was 5000 µg/plate.
The highest tested concentration in the mutation experiment 2 was selected based on the toxicity of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix; at 10 µg/plate (TA100)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without S9-mix; at 10 µg/plate (TA98)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix; at 4.0 µL/plate (TA102)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracine

Remarks:

With S9-mix; at 2.5 µg./plate (TA98 and TA100); at 10 µg/plate (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Experiment I) ; preincubation (Experiment II)
- Plate incorporation (Experiment I):
In the plate incorporation assay, the following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control); 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation); 100 µL Bacteria suspension (cf. test system, pre-culture of the strains); 2000 µL Overlay agar. After solidification the plates will be incubated upside down for at least 48 hours at 37° C in the dark.
- Pre-incubation (Experiment II):
In the pre-incubation method, 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates will be incubated upside down for at least 48 hours at 37° C in the dark.

DURATION
- Preincubation period: 60 minutes (Experiment II)
- Exposure duration: at least 48 hours (Experiments I and II)
- Selection time (if incubation with a selection agent): at least 48 hours (Experiments I and II) simultaneous with exposure duration
- Fixation time (start of exposure up to fixation or harvest of cells): at least 48 hours

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects of the test substance were detected by a substantial reduction in mean revertant colony counts or by the sparse or absent background bacterial lawn.

Rationale for test conditions:

Solubility limitations: Since the test item was fully soluble in DMSO, the highest tested concentration for the Mutation assay was 5000 μg/plate.

Evaluation criteria:

A test substance was considered a mutagen if a biologically relevant increases in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control was observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 98, TA 100, and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains: at 333 µg/plate and above (without S9-mix), and at 1000 µg/plate and above (with S9-mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA 98, TA 100, and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains: at 1660 µg/plate and above with and without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation:
Pre-Experiment/Experiment I: at 2500 µg/plate and above with S9-mix in all strains;
Experiment II: no precipitation observed up to the highest tested concentration (5000 µg/plate)
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I since the criteria mentioned above were met and 5000 µg/plate were chosen as maximum concentration. Based on the results of experiment I, 5000 μg/plate was selected as the highest test item concentration for experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Vehicle control: The vehicle control values were within the laboratory's historical control range.
- Positive control: Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects, evident as a reduction in the number of revertants, occurred with all strains with and without metabolic activation at the following concentrations: Experiment I - without S9 mix: 333-5000 µg/plate and with S9 mix: 1000-5000 µg/plate; Experiment II - with and without S9 mix: 1660 and 5000 µg/plate.

Remarks on result:

other: Experiment I

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
It was concluded that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.