t-Butyl (2S,2aS,5aR)-1-azabicyclo[3.3.0] oct-2-carboxylate monooxalate salt

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:

2006-09-28 to 2007-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) without deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to other 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

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from "The Department of Health of the Government of the United Kingdom"

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material: VRT-126028; (1S, 3aR, 6aS) tert-buty octahydrocyclopenta[c]pyrrole-1-carboxylic acid oxalate
- Molecular formula: C14H23NO6
- Molecular weight: 301.34
- Smiles notation: not applicable
- InChl: not applicable
- Structural formula attached as image file: not applicable
- Substance type: active
- Physical state: white powder
- Analytical purity: 99.3 % area by GC
- Impurities: no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 2006-05-22
- Lot/batch No.: batch 25414 and lot WYJ11410404533
- Expiration date of the lot/batch: 2008-05
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other: The test substance was received on 2006-09-15.

Method

Target gene:

histidine locus (S. typhimurium strains); tryptophan locus (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

other: Please see below for additional strain characteristics.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

other: Please see below for additional strain characteristics.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from rats with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Experiment 1 (with and without metabolic activation for all tester strains)-5, 15, 50, 150, 500, 1500 and 5000 ug/plate
Experiment 2 (with and without metabolic activation for all tester strains)-50, 150, 500, 1500 and 5000 ug/plate
The highest concentration tested in the study (5000 ug/plate) is the standard limit concentration recommended in the regulatory guidelines that this assay followed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solubility of the test substance was assessed at 50 mg/mL in water, in which it dissolved. Water (purified in-house by reverse osmosis and sterilized by autoclaving) was, therefore, used as the vehicle for the study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene 5 ug/plate for TA100 and TA1535 and 10 ug/plate for E. coli WP2 uvrA (pKM101)

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activiation

Migrated to IUCLID6: 5 ug/plate for TA98 and TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Migrated to IUCLID6: 2 ug/plate for TA100 and TA1535

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Migrated to IUCLID6: 50 ug/plate for strain TA1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Migrated to IUCLID6: 2 ug/plate for strain TA98

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide: 2 ug/plate for strain WP2 uvrA (pKM101)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1-in agar (plate incorporation); experiment 2-preincubation


DURATION
- Preincubation period: 30 minutes (experiment 2)
- Exposure duration: 72 hours (experiments 1 and 2)
- Expression time: not applicable
- Selection time: 72 hours (experiments 1 and 2); simultaneous with exposure
- Fixation time: not applicable


SELECTION AGENT:
- histidine (S. typhimurium strains) and tryptophan (E. coli strain)
SPINDLE INHIBITOR:
- not applicable
STAIN:
- not applicable


NUMBER OF REPLICATIONS:
- 3 (experiments 1 and 2)


NUMBER OF CELLS EVALUATED:
- not applicable


DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects of the test substance were detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn.


OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable


OTHER:
- Additional plates were prepared with the same methods as all of the other plates in the assay (experiment 1), except the addition of bacteria was not included. These plates were used to assess the sterility of the test substance, S9 mix and sodium phosphate buffer.

Evaluation criteria:

If exposure to the test substance produced a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it was considered to exhibit mutagenic activity in the test system.
If exposure to the test substance did not produce a reproducible increase in revertant colony numbers, it was considered to show no evidence of mutagenic activity in the test system.
If the results obtained failed to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data, may have been subjected to analysis to determine the statistical significant of any increase in revertant colony numbers. See statistics section below. Biological importance was considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls were not considered biologically important. It was acceptable to conclude an equivocal response if no clear results could be obtained.
If these criteria were not appropriate to the test data, the Study Director would use scientific judgement.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The "fold-increases" relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups. No statistical analysis was performed for tests judged as negative or positive. However, if tests failed to satisfy the criteria for a clear positive or negative response and statistical tests were deemed necessary, the statistical procedures used were those described by Mahon et al (1989) and were usually Dunnett's test followed , if appropriate, by trend analysis.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

experiments 1 and 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

experiments 1 and 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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: the test substance in water up to the stock concentration used in the study of 50 mg/mL
- Precipitation: no data
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES:
No range-finding test was performed. However, experiment 1 was used to specify the concentrations to be used in experiment 2.


COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle control were within or close to the 99% confidence limits of the current historical control range of the laboratory (experiments 1 and 2). Appropriate positive control substances (experiments 1 and 2) induced substantial increases (all within historical positive control range) in revertant colony numbers with all strains in all tests, confirming sensitivity of the cultures and activity of the S9 mix. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration up to 5000 ug/plate in either the presence or absence of metabolic activation in either of the experiments.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No evidence of toxicity was obtained following exposure to the test substance in either of the experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The absence of colonies on sterility check plates (experiment 1) confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation. Total colony counts on nutrient agar plates (100 uL aliquots of E-6 dilution of 10 -hour bacterial culture) confirmed the viability and high cell density of the cultures of the individual organisms.

Applicant's summary and conclusion

Conclusions:

The test substance was evaluated for mutagenic potential using the Ames assay in S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA (pKM101) both in the presence and absence of metabolic activation. It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed up to a concentration of 5000 ug/plate.

Executive summary:

Not applicable