N-phenyl-N-piperidin-4-ylpropionamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-06-10 to 2005-06-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non-GLP study performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay) with the following deviations: 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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only three strains of bacteria were used.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D, dated May 19, 2000

Deviations:

yes

Remarks:

Only three strains of bacteria were used.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-phenyl-N-piperidin-4-ylpropionamide

EC Number:

216-543-3

EC Name:

N-phenyl-N-piperidin-4-ylpropionamide

Cas Number:

1609-66-1

Molecular formula:

C14H20N2O

IUPAC Name:

N-phenyl-N-piperidin-4-ylpropionamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-1594255-AAA (T000425)
- Physical state: solid (powder)
- Appearance: white

Specific details on test material used for the study:

Description: white solid
Batch number: 00454795
Purity: 100 %
Stability in solvent: not indicated by sponsor
Solubility in water: 43 g/L
Solubilityin ethanol: 500 g/L
Storage conditions: Room temperature
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/beta-naphtoflavone induced rate liver S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

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 metabolic activation (S9): for TA100 at 10 μg/plate

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 metabolic activation (S9): for TA98 at 10 μg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

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

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

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 and IIa: 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 (mutation assays): histidine (S. typhimurium strains)

NUMBER OF REPLICATIONS: triplicates

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

OTHER: The pre-experiment was reported as main experiment I because no toxic effects were observed.
Due to irregular bacteria growth of strain TA98 in experiment II, this part of experiment had to be repeated.

Evaluation criteria:

A test substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice(strains TA98, TA100, and TA102) 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 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:

no 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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: 43 g/L
- Precipitation: No precipitation of the test substance in the overlay agar was observed.

RANGE-FINDING/SCREENING STUDIES:
The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

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.