Reaction mass of (2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide and (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

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:

13 September 2006 - 3 November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide; (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

EC Number:

700-041-0

Molecular formula:

C11 H20 N2 O2

IUPAC Name:

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide; (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

Test material form:

solid: particulate/powder

Remarks:

white powder

Details on test material:

- Name of test material (as cited in study report): ucb108628-1
- Stability under test conditions: not indicated
- Storage condition of test material: room temperature, in the dark

Method

Target gene:

Histidine gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone (80/100 mg per kg per day) induced rat liver S9 mix

Test concentrations with justification for top dose:

Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
First mutation test: 50, 150, 500, 1500 and 5000 μg/plate
Second mutation test: 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Solvent used: sterile distilled water
- Justification for choice of solvent/vehicle: The test material was soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

without S9

Controls 2

Positive controls:

yes

Remarks:

without S9

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

3 μg/plate for TA100 and 5 μg/plate for TA1535

Controls 3

Positive controls:

yes

Remarks:

without S9

Positive control substance:

9-aminoacridine

Remarks:

80 μg/plate for TA1537

Controls 4

Positive controls:

yes

Remarks:

without S9

Positive control substance:

mitomycin C

Remarks:

0.5 μg/plate for TA102

Controls 5

Positive controls:

yes

Remarks:

without S9

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 μg/plate for TA98

Controls 6

Positive controls:

yes

Remarks:

with S9

Positive control substance:

other: 2-aminoanthracene

Remarks:

1 μg/plate for TA100 and 2 μg/plate for TA1535 and TA1537

Controls 7

Positive controls:

yes

Remarks:

with S9

Positive control substance:

benzo(a)pyrene

Remarks:

5 μg/plate for TA98

Controls 8

Positive controls:

yes

Remarks:

with S9

Positive control substance:

other: 1,8-dihydroxyanthraquinone

Remarks:

10 μg/plate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: in triplicate against each tester strain

DETERMINATION OF CYTOTOXICITY
- Method: numbers of revertant colonies and growth of the bacterial background lawn

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100).

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. A small, statistically significant increase (P≤0.05) in revertant colony frequency was observed in tester strain TA1535 (without S9) at 5000 μg/plate in Experiment 1 only. This increase was within the spontaneous mutation range and the historical range expected for this strain, was non-reproducible and exhibited no dose response relationship. Therefore, the increase was considered to be of no toxicological relevance.

Applicant's summary and conclusion

Conclusions:

An in vitro gene mutation study (AMES study) was conducted according to OECD/EC guidance and GLP principles. The test substance was found to be non-mutagenic under the conditions of this test.