1-amino-N-[6-cyano-5-(trifluoromethyl)-3-pyridyl]cyclobutanecarboxamide

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 2016-02-23 to 2016-03-11

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:

Adopted 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-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Cas Number:

1950587-17-3

Molecular formula:

C12H11F3N4O

IUPAC Name:

1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: JNJ-63632283-AAA (T003665)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:I15HD3050
- Expiration date of the lot/batch: 2017-08-17
- Physical Description: White powder
- Purity: 98.1 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: store from +15°C to 25°C
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the vehicle: Not indicated

OTHER SPECIFICS
- Correction factor: 1

Method

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

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 body weight).
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution ; 1 mL 0.33 M KCl solution. 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.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% (v/v) S9-mix in the first experiment and 10% S9-mix (v/v) in the second experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch is characterized 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:

The maximum final concentration for the dose range finding test was selected based on the solubility of the test item in Dimethyl sulfoxide (highest concentration recommended in OECD test guideline).
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 with and without 5% (v/v) S9-mix.

The top doses for the mutation experiments were selected based on the solubility of the test item observed in the dose range finding test.
Mutation experiment 1: 52, 164, 512, 1600 and 5000 μg/plate in TA1535, TA1537, TA98 and TA102 with and without 5% (v/v) S9-mix
Mutation experiment 2: 154, 275, 492, 1000, 1400 and 2000 μg/plate in all tester strains with and without 10% (v/v) S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)

- Justification for choice of solvent/vehicle:
The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml. Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix; 5 μg/plate (TA1535)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9-mix; 2.5 μg/plate (TA1537)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix; 10 μg/plate (TA98)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix; 650 μg/plate (TA100)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: tert-butyl hydroperoxide (TBH)

Remarks:

Without S9 mix , 250µg (TA102)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix; 2.5 μg/plate (TA1535 at 5 and 12.5% S9; TA1537 at 5% S9), 5 μg/plate (TA153 7 at 12.5% S9), 1 μg/plate (TA98 at 5 and 12.5% S9; TA100 at 5% S9), 2 μg/plate (TA100 at 10% S9), 10 μ g/plate (TA102 at 5 and 12.5% S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- 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 (10^9 cells/mL) of one of the tester strains,
- 0.1 mL of a dilution of the test item in DMSO or Milli-Q water 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

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

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three 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 the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle 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.

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:

exp 1 (absence of S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

exp 1 (absence of S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp 1 and exp 2 (absence of S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp 1 & 2 (absence and presence of S9-mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility:The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the tester strains TA100 at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537, TA98 and T102 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000μg/plate.


STUDY RESULTS
- Concurrent vehicle negative and positive control data: 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.


- Precipitate:
- Mutation Experiment 1:Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and no precipitate was observed at the end of the incubation period.
- Mutation Experiment 2: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and no precipitate was observed at the end of the incubation period. Except in tester strain TA102 where no precipitation of the test item on the plates was observed at the start of the incubation period.

- Signs of toxicity:
- Mutation Experiment 1: No reduction of the bacterial background lawn was observed in any of the conditions tested.
In the absence of S9-mix, moderate reduction in the number of revertant colonies was observed at 5000 µg/plate for TA1535 and TA98. Slight and extreme reductions was observed for TA102 at 1600 and 5000 µg/plate, respectively.
In the presence of S9-mix, a moderate reduction in the number of revertant colonies was observed at 5000 µg/plate for TA102.
All other conditions, not mentioned here, showed no biologically relevant reduction in the number of revertant colonies.
In strain TA1537 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. Since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely that these reductions are caused by incidental fluctuations in the number of revertant colonies.

- Mutation Experiment 2: No reduction of the bacterial background lawn was observed in any of the conditions tested.
In the absence of S9-mix, moderate reduction in the number of revertant colonies was observed at 5000 µg/plate for TA98. Slight, moderate and extreme reductions were observed for TA102 at 1568, 2800 and 5000 µg/plate, respectively.
In the presence of S9-mix, slight and extreme reductions in the number of revertant colonies were observed at 2800 and 5000 µg/plate for TA102, respectively.
All other conditions, not mentioned here, showed no biologically relevant reduction in the number of revertant colonies.
In strain TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. Since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely that these reductions are caused by incidental fluctuations in the number of revertant colonies.

- 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.

- 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 two experiments.
The vehicle control values were within the laboratory historical control data ranges, except the response for TA1535 in the first experiment (absence of S9-mix). Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (4 relevant colonies) when compared against relevant historical control data (5 relevant colonies), the validity of the test was considered to be not affected.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay.