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

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:

2005-05-12 to 2005-08-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only three strains are tested. However, an expert statement is added in the field "any other remarks" to justify the fact that no further testing is necessary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Only 3 tester strains were used.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 3 tester strains were used.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

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: white solid
- Analytical purity: 100 %
- Impurities: no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: batch 9852
- Expiration date of the lot/batch: 2005-12-31
- Stability under test conditions: no data
- Storage condition of test material: room temperature, protected from light
- Other: solubility in water = 0.13 g/L; solubility in ethanol >500 g/L

Specific details on test material used for the study:

Description: white solid
Batch number:RT000424G1A251
Purity: 100 %
Stability in solvent: Not indicated by sponsor
Solubility in water: 0.13 g/L
Solubilityin ethanol: > 500 g/L
Storage conditions: Room temperature, protected from light
Expiry date: 2005-12-31

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100 and TA102

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 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- 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

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: sodium azide

Remarks:

Without metabolic activation (S9): for TA100 at 10 μg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation (S9): for TA98 at 10 μg/plate

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (S9): for TA102 at 4 μg/plate

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation (S9): for all strains, 2.5 μg/plate for strains TA98 and TA100 and 10 μg/plate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) (pre-experiment, experiment I); preincubation (experiment II).

Experiment I: in agar (plate incorporation)
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 (test system, pre-culture of the strains);
- and 2000 μL overlay agar.

Experiment II: preincubation
- In the pre-incubation assay, 50 μL test solution, solvent or 100 μL positive control, 500 μL S9 mix/S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation, 2.0 mL overlay agar (45°C) was added to each tube. The mixture
was poured on minimal agar plates.
- After solidification, the plates from both assays were 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
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

The test substance was considered a mutagen if a biologically relevant increase 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 remained within the historical range of negative and solvent control such an increase was not considered biologically relevant.

Statistics:

A statistical analysis of the data was not required.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100 and TA102

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
- Evaporation from medium: no data
- Water solubility: 0.13 g/L in water
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:
Experiment 1 was used as the range-finding test. See relevant sections

COMPARISON WITH HISTORICAL CONTROL DATA:
Appropriate reference mutagens were used as positive control. They showed a distinct increase in induced revertant colonies.The positive control mean values in experiment 1 for strains TA100 and TA102 were slightly above the max values in the historical data.The positive control mean value in experiment 2 for strain TA98 was slightly above the max value in the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see below

Remarks on result:

other: strain/cell type: TA98, TA100, TA102

Any other information on results incl. tables

Table 2. Reduced background growth was observed at the following concentrations (ug/plate)
Strain	Experiment 1		Experiment 2
	Without S9	With S9	Without S9	With S9
TA98	2500-5000	2500-5000	1000-5000	1000-5000
TA100	2500-5000	2500-5000	1000-5000	1000-5000
TA102	/	2500-5000	1000-5000	1000-5000
Toxic effects, evident as a reduction in the number of revertants were observed at the following concentrations (ug/plate)
Strain	Experiment 1		Experiment 2
	Without S9	With S9	Without S9	With S9
TA98	2500-5000	2500-5000	1000 -5000	1000-5000
TA100	/	2500-5000	2500-5000	2500-5000
TA102	5000	2500-5000	1000-5000	1000-5000

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was assessed for its potential to induce gene mutations according to the plate incorporation test and the pre-incubation test using Salmonella typhimurium strains TA98, TA100 and TA102 in the presence and absence of metabolic activation. Based on the results of the study, it was concluded that the test substance was considered negative for mutagenic potential (base pair changes and frameshift mutations) in the test system.