1-[(tert-butoxycarbonyl)amino]cyclobutane-1-carboxylic acid

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:

2015-12-07 to 2015-12-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

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:

1-[(tert-butoxycarbonyl)amino]cyclobutane-1-carboxylic acid

EC Number:

627-199-2

Cas Number:

120728-10-1

Molecular formula:

C10H17NO4

IUPAC Name:

1-[(tert-butoxycarbonyl)amino]cyclobutane-1-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

Physical state: powder
Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15BD0548
- Expiration date of the lot/batch: 2017-01-30 (retest date)
- Appearance: White powder
- Purity: 94.0% (w/w)
- Purity test date: 2015-09-11

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, dessicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test item was dissolved in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany).

OTHER SPECIFICS:
- Correction factor: 1.06
- Test item concentrations were used within 3 hours of preparation.

Method

Target gene:

histidine locus (histidine-dependent S. typhimurium strains)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg).
- Method of preparation of S9 mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml (first and third experiment) or 5.0 ml Milli-Q water (second experiment) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first and third experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9- fraction) to complete the S9-mix in the second experiment.
- Concentration or volume of S9 mix and S9 in the final culture medium: 5% (v/v) S9-mix in the first experiment, 10% S9-mix (v/v) in the second experiment and 5% (v/v) S9-mix in the third experiment.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch is characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

Dose-range finding test: 1.6, 5.1, 16, 49, 155, 483, 1509 and 4717 µg/plate with and without 5% S9-mix (TA100).
Mutation experiment I: 49, 155, 483, 1509 and 4717 µg/plate with and without 5% S9-mix (TA1535, TA1537, TA98 and TA102).
Mutation experiment II: 492, 878, 1568, 2800 and 5000 µg/plate with and without 10% S9-mix (all tester strains).
Mutation experiment III: 2500 and 5000 µg/plate with and without 5% S9-mix (all tester strains).

Since the test item was soluble in DMSO at 47.17 mg/ml (=4717 µg/plate), this was selected as the highest dose level for the dose range finding test. Based on the results of the dose range finding test, 4717 and 5000 µg/plate were selected as the highest dose levels for the mutation experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 47.17 mg/ml. In DMSO, the test item was soluble at 47.17 mg/ml (= 4717 µg/plate). Based on these solubility findings, DMSO was selected as vehicle and 4717 µg/plate was selected as the maximum final concentration for the dose range finding test.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation; 5 μg/plate (TA1535)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without metabolic activation; 2.5 μg/plate (TA1537)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation; 10 μg/plate (TA98)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; 650 μg/plate (TA100)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: tert-butyl hydroperoxide

Remarks:

Without metabolic activation; 250 μg/plate (TA102)

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix; 2.5μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2μg/plate (TA100 with 10% S9-mix), 10μg/plate (TA102 with 5 and 10% S9-mix)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 3

METHOD OF APPLICATION:
- test substance added in agar (plate incorporation)
- Method: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 h

SELECTION AGENT (mutation assays): histidine


METHODS FOR MEASUREMENT OF CYTOTOXICITY:
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and/or the reduction of the revertant colonies

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done. In addition to the criteria stated below, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate in TA1535 without S9-mix (experiment 3)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

at 4717 µg/plate in TA102 without S9-mix (experiment 1), at 4717 µg/plate in TA102 with and without S9-mix (experiment 2) and at 2500 and 5000 μg/plate with and without S9-mix (experiment 3)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Selection of an adequate range of doses was based on a dose range finding test with tester strain TA100 with and without 5% (v/v) S9-mix. Eight concentrations, 1.6, 5.1, 16, 49, 155, 483, 1509 and 4717 µg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation experiment was 4717 µg/plate. Based on the results, the results of the dose range findings are part of the first experiment and the following dose range was selected for the first mutation experiment with the tester strains, TA1535, TA1537, TA98 and TA102 in the absence and presence of S9-mix: 49, 155, 483, 1509 and 4717 μg/plate.

STUDY RESULTS
- Precipitate:
- Mutation Experiment 1: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Mutation Experiment 2: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
- Mutation Experiment 3: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

- Signs of toxicity:
- Mutation Experiment 1: A slight reduction in the number of revertants was observed in tester strain TA102 (absence of S9-mix) at the highest dose level tested. In all other strains, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA100 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed and the reductions were less than 20% compared to the concurrent vehicle control, these reduction are not considered to be caused by toxicity of the test item, and rather it is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
- Mutation Experiment 2: Reductions in the number of revertant colonies below the laboratory historical control data range were observed in tester strain TA102 in the absence of S9-mix. At the dose levels of 878 to 2800 µg/plate the reduction was less than 20% compared to the concurrent vehicle control, whereas at the top dose of 5000 µg/plate a slight reduction in the number of revertants was observed compared to the concurrent vehicle control. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix
- Mutation experiment 3: In tester strain TA102, in the absence and presence of S9-mix, reductions in the number of revertant colonies below the laboratory historical control data range were observed. At the dose level of 2500 µg/plate in the presence of S9-mix the reduction was less than 20% compared to the concurrent vehicle control, whereas at the dose levels of 2500 and 5000 µg/plate in the absence of S9-mix and at the top dose of 5000 µg/plate in the presence of S9-mix slight reductions in the number of revertants were observed compared to the concurrent vehicle control. In tester strain TA1535, in the absence of S9-mix, a slight reduction in the number of revertants was observed at 5000 µg/plate compared to the concurrent vehicle control. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the other tester strains in the absence and presence of S9-mix.

- Mutagenicity:
- Mutation Experiment 1: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
- Mutation Experiment 2: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested
- Mutation Experiment 2: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested

- Discussion: All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments.

HISTORICAL CONTROL DATA:
- The negative control values were within the laboratory historical control data ranges, except the response for TA100 in the first experiment. Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (54 and 65 revertant colonies in the absence and presence of S9-mix, respectively) when compared against relevant historical control data (64 and 70 relevant colonies in the absence and presence of S9-mix, respectively), the validity of the test was considered to be not affected.
- The 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, except the responses for TA102 (absence of S9-mix, second and third experiment; presence of S9-mix, first experiment) . The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the values were 5.7 times greater in the presence of S9-mix and 1.7 to 2.1 times greater in the absence of S9-mix than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the validity of the test results.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay.