1-benzyl-N-phenylpiperidin-4-amine

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:

2006-03-29 to 2006-04-20

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:

2006

Report date:

2006

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 tester 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 tester strains were used.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-benzyl-N-phenylpiperidin-4-amine

EC Number:

214-583-6

EC Name:

1-benzyl-N-phenylpiperidin-4-amine

Cas Number:

1155-56-2

Molecular formula:

C18H22N2

IUPAC Name:

1-benzyl-N-phenylpiperidin-4-amine

Details on test material:

- Name of test material (as cited in study report): N-phenyl-1-(phenylmethyl)-4-piperidinamine (T000293)
- Substance type: no data
- Physical state: white solid
- Analytical purity: 100 %
- Lot/batch No.: batch 00479757 RT000293G1A401
- Expiration date of the lot/batch: 2006-06-30
- Stability in solvent: not indicated by the sponsor
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

Description: white solid
Batch number: 00479757 RT000293G1A401
Purity: 100 %
Stability in solvent: not indicated by sponsor
Storage conditions: Room temperature
Expiry date: 2005-06-30

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-naphthoflavone induced rat 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: 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

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation; at 10 µg/plate for TA100

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation; at 10 µg/plate for TA98

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation; at 4.0 µL/plate for TA102

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation; at 2.5 µg/plate for TA98 and TA100 and 10.0 µg/plate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In Experiment 1 (plate incorporation), 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 S9) or S9 mix substitution buffer (for test without S9),
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL overlay agar.

In Experiment 2 (pre-incubation), 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°C) 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
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicates

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 as 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 controls such an increase was not considered biologically relevant.

Statistics:

A statistical analysis of the data was not required according to guideline

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
- Water solubility: no data
- Precipitation: Precipitation of the test substance was observed in the test tubes in the overlay agar from 2500 µg/plate up to 5000 µg/plate. The undissolved particles of the test substance had no influence on the data recording.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- The pre-experiment was reported as main Experiment 1. See relevant sections for results.

COMPARISON WITH HISTORICAL CONTROL DATA:
- In Experiment 1, the positive controls for TA98 in the presence and absence of metabolic activation, the positive control for TA100 in the presence of metabolic activation and the negative control for TA102 in the absence of metabolic activation produced mean revertant values slightly above historical data control range.
- In Experiment 2, the positive control for TA98 in the absence of metabolic activation produced a mean revertant value slightly above the historical data control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The plates incubated with the test substance showed reduced background growth at higher concentrations in both experiments with and without S9 mix (Table 1).

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

The test substance was evaluated for mutagenic potential in the bacterial reverse mutation assay in S. typhimurium strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation. Under the conditions of the study, the test substance was determined to be negative for mutagenic potential in all tester strains in the absence and presence of metabolic activation.