Ethyl 4-oxopiperidine-1-carboxylate

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:

from 2006-04-26 to 2006-05-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well documented study performed according to OECD guideline 471. No deviations were recorded. Expert statement on use of 3 strains is added in attachment and in the field "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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 4-oxopiperidine-1-carboxylate

EC Number:

249-984-5

EC Name:

Ethyl 4-oxopiperidine-1-carboxylate

Cas Number:

29976-53-2

Molecular formula:

C8H13NO3

IUPAC Name:

ethyl 4-oxopiperidine-1-carboxylate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study reports): JNJ-126971-AAA (T000509)
- Physical state: liquid
- Appearance: clear colourless to light yellow liquid

Specific details on test material used for the study:

Batch Number: 00451179 HT000509G3I381
Aggregate state at room temperature: liquid
Colour: colourless to light yellow
Purity: 100%
Stability in solvent: not indicated by the sponsor
Storage: Room temperature
Expiry date: 2008-09-21

Method

Target gene:

The histidine dependent strains were derived from S. typhimurium strain LT2 through a mutation in the histidine locus.

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-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.

5000 µg/plate is the maximum dose level to be tested in the absence of toxic effects.

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation, 10 µg/plate, strain: TA 100

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation, 10 µg/plate, strain: TA98

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, 4.0 µL/plate, strain: TA 102

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation, 2.5 µg/plate (TA 98, TA 100), 10 µg/plate (TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: plate incorporation; Experiment II: preincubation. Experimental performance: 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. In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken 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.

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: For each strain and dose level, including the controls three plates were used.

DETERMINATION OF CYTOTOXICITY
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is 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 is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.A dose dependent increase in the number of revertant colonies below the threshold is 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 is not considered biologically relevant.

Statistics:

A statistical analysis of the data was not required.

Results and discussion

Test results

Species / strain:

other: TA 98, TA 100 and TA 102

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
- Precipitation: No precipitation of the test item occured up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES:To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment 1 (plate incorporation test). 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 no toxic effects were observed six concentrations were tested in experiment II and 5000 µg/plate were chosen as maximum concentration.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants, occured in the test groups 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

In conclusion, it can be stated that during the described mutagenicity test and under the experminental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.