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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 October 2000 - 01 December 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
lacking experimental data; seeding density
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
(2S)-2-(2-oxo-4R-propylpyrrolidin-1-yl)butanamide
Cas Number:
357336-20-0
IUPAC Name:
(2S)-2-(2-oxo-4R-propylpyrrolidin-1-yl)butanamide
Test material form:
solid: particulate/powder
Details on test material:
- Description: White powder
- Storage conditions: At ambient temperature in the dark

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254-induced S9 enzymes (the supernatant of the post-mitochondial 9000 g fraction) were prepared from the livers of adult, male Fischer rats
- method of preparation of S9 mix: according to McGregor et al (1988): 'Studies of an S9-based metabolic activation system used in the mouse Iymphoma L5178Y cell mutation assay'. Mutagenesis, 3, 485-490.
- concentration or volume of S9 in S9 mix: 10% (v/v)
- quality controls of S9: the enzymic activity of the batch of S9 was characterised by testing pre-mutagens using TA 1538.
Test concentrations with justification for top dose:
- Toxicity test, with TA 100 only (with and without S9 mix): 0.1, 1, 10, 100, 1000 and 5000 µg/plate
- Main test 1, all strains (with and without S9 mix): 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate
- Main test 2, all strains (with and without S9 mix): 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate

All handling of the test substance and its solutions was conducted under amber light. The formulations were prepared within 1 hour of dosing.
Vehicle / solvent:
- Vehicle used: Dimethylsulphoxide (DMSO)
- Justification for choice of vehicle: Solubility tests showed that at 50 mg/mL, the test substance was more soluble in this vehicle than in water
- For testing a stock solution of the test substance at 50 mg/mL was prepared
- This stock solution was subsequently passed through a 0.2 µm filter.
Evaluation: Considered not to have influenced the study. The solubility of the registered substance was shown to be high in DMSO (in the skin sensitization study for instance).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See the section 'any other information on materials and methods'
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS
- Number of cultures per concentration: toxicity test: single; main tests: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE
- Cell density at seeding: no data
- Preincubation

TREATMENT AND HARVEST SCHEDULE
- Preincubation period, if applicable: 20 min. at 37°C
- Exposure duration/duration of treatment: 48-72 hours at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background lawn growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutagenic response was evaluated by the increase in the number of revertant colonies.

Evaluation criteria:
A test was considered acceptable, if for each strain:
i) the bacteria demonstrated their typical responses to crystal violet, ampicillin and U.V.light.
ii) the vehicle control mutant colony counts were within or close to the historical ranges for the accumulated laboratory database.
iii) there were at least 2-fold increases over the mean vehicle control values in at least 2 of the positive control plates for each strain and activation state (in the case of TA 100, 1.5-fold was required).
If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
iv) in at least the second mutation experiment, no toxicity or contamination was observed in at least 5 dose levels.
v) in cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
i) for S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E.coli, at least a doubling of the mean concurrent vehicle control values at some concentration of the test material. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value was considered significant. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 was required before a significant mutagenic response was registered.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolizing enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.
Statistics:
Not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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
Remarks:
of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed up to and including a concentration of 5000 µg/plate with and without S9-mix.

RANGE-FINDING/SCREENING STUDIES: No toxicity to the bacteria was observed and no precipitation of the test item occurred in either activation condition.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The vehicle control values were generally within the normal ranges experienced in the laboratory and reported in the literature with these strains of S. typhimurium and E. coli, (Ames et al, 1975; Gatehouse et al, 1994).
The results obtained in the positive control groups were generally within the normal ranges experienced in this laboratory for each bacterial strain and activation condition, except in the retests of TA 1535 from the first and second mutation assays, where the counts obtained with ENNG were outwith the range presented in the historical data. For comparison, TA 1535 was also tested with Sodium azide at 1 µg per plate (in the absence of S9 mix) during the retest from the second mutation assay. The results obtained in this instance were satisfactory, yielding an average count of 314 colonies per plate. Also the counts obtained for TA 1535 with 2-AAN in the presence of S9 mix were acceptable on all occasions indicating that the low results obtained with ENNG were due to the popr performance of the positive control itself and not the strain.
In view of this, it was decided that the data was acceptable.

- Signs of toxicity : There was no toxicity to the bacteria.
- The substance did not induce mutagenic activity in any of the 5 bacterial strains used, in either activation condition. It was noted for strain TA 100 in the absence of S9 mix that the number of revertant colonies exceeded 1.5-fold the negative control value on some occasions (toxicity test, second mutation test). This was probably related to some rather low plate counts being obtained for the concurrent vehicle controls. The increases were small, however, there was no evidence of a dose-relationship and the effect was not reproducible. Moreover, the number of revertants did not exceed the historical negative control data. It was therefore considered that the increase was an artefact and not due to the test item.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
See the attached Hitstorical data_report 19346 in the section attached background material.

Applicant's summary and conclusion

Conclusions:
In an Ames test, performed equivalent to OECD 471 guideline and GLP principles, the substance was found not to be mutagenic with or without metabolic activation.
Executive summary:

An Ames test was performed equivalent to OECD 471 guideline and GLP principles.

In the dose-range finding study, the test item was tested up to and including concentrations of 5000 μg/plate in strain TA100 in the pre-incubation assay. The substance did not precipitate on the plates at this dose level and the bacterial background lawn was not reduced at any of the concentrations tested.

In the mutation experiments, the test item was tested up to and including concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level and no cytotoxicity was observed.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in independently repeated experiments. In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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