4-Bromo-2-fluoro-N-methylbenzamide

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 2016-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21July 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:

4-Bromo-2-fluoro-N-methylbenzamide

EC Number:

808-257-8

Cas Number:

749927-69-3

Molecular formula:

C8H7BrFNO

IUPAC Name:

4-Bromo-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: M15BD0474
- Expiration date of the lot/batch: 2017-01-15
- Physical Description: White powder with lumps
- Purity: 99.8%
- Purity test date: 2015-09-11


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
-The test item was dissolved in dimethyl sulfoxide.

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: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 or 5.0 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 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 in the absence and presence of S9-mix. The highest concentration of the test item used in the subsequent mutation experiment was 5000 µg/plate.

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: 312.5, 625, 1250, 2500 and 5000 μg/plate in all tester strains with and without 10% (v/v) S9-mix in all tester strains.

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 (= 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: tert-butyl hydroperoxide

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:

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

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:

cytotoxicity

Remarks:

Experiment 1

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 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2

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 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Experiment 2

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1&2

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1&2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1&2

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. 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 TA102 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 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 TA102 in the first and second experiment (absence of
S9-mix) and TA1535 in the second experiment (absence of S9-mix). Since the mean number of revertant colonies showed a characteristic number of revertant colonies (246 and 245 for TA102, 4 for TA1535) when compared against relevant historical control data (248 and 5 relevant colonies, 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 TA100 (dose range finding test, presence of S9-mix) and TA102 (first experiment, absence of S9-mix). The positive control is included 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 5.6 and 1.7-times greater than the concurrent solvent control value, for TA100 and TA102 respectively, 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 not observed 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.

- Signs of toxicity:
- Mutation Experiment 1: Reduction of the bacterial background lawn and biologically relevant decrease in the number of revertants were observed in all strains with or without S9.
- Mutation Experiment 2: Reduction of the bacterial background lawn and biologically relevant decrease in the number of revertants were observed in all strains except TA98 without S9. Reduction of the bacterial background lawn and biologically relevant decrease in the number of revertants were observed in all strains except TA98 and TA1535 with S9. In tester strain TA1535 with S9, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid dose of 625 µg/plate. Since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test substance, rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies. 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 tester strain TA98 with or without S9.

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: In the second mutation assay, 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.

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.