[No public or meaningful name is available]

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 2015-12-14 to 2016-01-14

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:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”.

Version / remarks:

Official Journal of the European Union No. L142, 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:

4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide

Cas Number:

1950587-20-8

Molecular formula:

C20H17F4N5O2

IUPAC Name:

4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: I15DD1622
- Expiration date of the lot/batch: 2017-04-15
- Physical Description: Brown crytalline powder with small clots
- Purity: 103.0 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the vehicle: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test item was dissolved in dimethyl sulfoxide (DMSO). Preparation of test solutions started with solutions of 50 mg/ml applying treatment with ultrasonic waves resulting in a clear brown solution.

FORM AS APPLIED IN THE TEST: liquid


OTHER SPECIFICS
- Correction factor: 1

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

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:
- First experiment: 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.
-Second experiment: 37.5 mg NADP and 19 mg glucose-6-phosphate in 6.25 ml Milli-Q water; 1.25 ml 0.5 M sodium phosphate buffer pH 7.4; 0.63 ml 0.08 M MgCl2 solution; 0.63 ml 0.33 M KCl solution. To 8.75 ml of S9-mix components 1.25 ml S9-fraction was added (12.5% (v/v) S9-fraction) to complete the S9-mix (deviation 2).

- 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 DMSO (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:
Absence of S9- mix: 1.7, 5.4, 17, 52, 164, and 512 μg/plate in TA1535, TA1537, TA98, TA100 and TA102
Presence of S9mix: 0.18, 0.55, 1.7, 5.4, 17, 52, 164 and 512 μg/plate in TA1535, TA1537, TA98, TA100 and TA102
Mutation experiment 1A: 512, 1600 and 5000 μg/plate in TA1535, TA1537, TA98, TA100 and TA102 with and without S9-mix
Mutation experiment 2: 275, 492, 878, 1568, and 2800 μg/plate in all tester strains with and without 12.5% (v/v) S9-mix

Vehicle / solvent:

- Vehicle(s): 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 (= 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.

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:

Remarks:

Without S9-mix; 250 μg/plate (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:

Wirh 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 12.5% S9), 10μ g/plate (TA102 at 5 and 12.5% S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 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 (109 cells/mL) of one of the tester strains,
- 0.1 mL to 0.3 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 (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) 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 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.

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:

no cytotoxicity, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

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, but tested up to precipitating 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:

no cytotoxicity, but tested up to precipitating concentrations

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
- Water 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 tester strain 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. Since precipitation of the test item was observed in almost all concentrations tested, tester strain TA100 was again tested in the first mutation experiment. Based on the results of the dose range finding test, the following dose ranges were selected for the first mutation experiment with the tester strains, TA1535, TA1537, TA98, TA100 and TA102:
Absence of S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate.
Presence of S9-mix: 0.18, 0.55, 1.7, 5.4, 17, 52, 164 and 512 μg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The negative control values were within the laboratory historical control data ranges, except the response for TA100 in the presence of S9-mix, dose range finding test. Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (65 revertant colonies) when compared against relevant historical control data (66 relevant colonies), 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 TA100 (dose range finding test, presence of S9-mix) and TA102 (additional first mutation experiment and second mutation experiment, absence of S9-mix). 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 6.8 times greater for TA100 and 1.5 times and 1.8 times for TA102 greater than the concurrent solvent control value, this deviation in the mean plate count of the positive control had no effect on the validity of the test results.

- Precipitate:
- Mutation Experiment 1:Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 164 μg/plate and 512 μg/plate. No precipitation of the test item on the plates was observed at the end of the incubation period.
- Mutation Experiment 1A: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards. At the end of the incubation period, precipitation of the test item on the plates was observed at concentrations of 1600 and 5000 μg/plate in the absence and presence of S9-mix.
- Mutation Experiment 2: Precipitation of the test item on the plates was observed at the start and the end of the incubation period at concentrations of 1568 and 2800 μg/plate. Except in tester strain TA1537 in the absence of S9-mix and TA1535 and TA100 in the presence of S9-mix, where precipitation at the end of the incubation period was only observed at the top dose of 2800 μg/plate.

- Signs of toxicity
:
- Mutation Experiment 1: A slight reduction in the number of revertant colonies was observed at the lowest dose of 1.7 μg/plate in the absence of S9-mix. Since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item, rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
First mutation experiment:
A slight reduction in the number of revertant colonies was observed in tester strain TA98 at the top dose of 512 μg/plate in the absence of S9-mix. In all other strains, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
- Mutation Experiment 1A: There was no reduction in 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 2:Slight reductions of the bacterial background lawn were observed at the highest tested concentration of 2800 μg/plate in tester strains TA1535, TA1537 and TA100 in the absence of S9-mix and in strains TA1535 and TA100 in the presence of S9-mix.
Furthermore a slight reduction of the bacterial background lawn was also observed at test concentrations of 1568 μg/plate in tester strain TA1535 in the absence of S9-mix.
In the other tester strains, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the absence and presence of S9-mix,
In strain TA102, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed in the absence of S9-mix at the top dose of 2800 μg/plate. However, since the reduction was less than 20% compared to the concurrent vehicle control, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation 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 1A: 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.

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.