3-bromo-5-fluoro-1-[(4-methylphenyl)sulfonyl]-1H-pyrrolo[2,3-b]pyridine

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:

08 September 2016 to 13 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study conducted according to OECD Guideline 471 and EC No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B. 13/14. No deviations were recorded.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: European Community (EC). Commission Regulation (EC) No. 440/2008, Part B.: Methods for the Determination of Toxicity and other health effects, Guideline B. 13/14

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:

3-bromo-5-fluoro-1-(4-methylbenzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine

Cas Number:

1417421-99-8

Molecular formula:

C14H10BrFN2O2S

IUPAC Name:

3-bromo-5-fluoro-1-(4-methylbenzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15ID3592
- Expiration date of the lot/batch: 21-09-2017
- Purity test date: 28-04-2016



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in tetrahydrofuran (THF; Hipersolv Chromanorm, VWR, Belgium)


OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied

Method

Target gene:

The Histidine locus (S. typhimurium histidine-dependent strains TA98, TA100, TA102, TA1535 and TA1537)

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor 1254 induced rat liver metabolic activation system)

Test concentrations with justification for top dose:

- Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate; The highest concentration of the test item used in the subsequent mutation assay was the level at which the test item exhibited limited solubility, 512 μg/plate in the first mutation experiment and 492 μg/plate in the second mutation experiment.
- Mutation experiment I: : 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Mutation experiment II: 48, 86, 154, 275 and 492 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: For Test Article: Tetra hydrofuran (THF)
For Positive Controls: DMSO and saline
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water, dimethyl sulfoxide and ethanol at 50 mg/ml. In THF, the test item was soluble at 100 mg/ml (= 5000 µg/plate). Based on these solubility findings, THF was selected as vehicle.

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), dissolved in Saline

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), dissolved in DMSO

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), dissolved in DMSO

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), dissolved in DMSO

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), dissolved in DMSO

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 metabolic activation, 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 μg/plate (TA102 at 5 and 10% S9), dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48+-4h

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium histidine-dependent strains)

NUMBER OF REPLICATIONS: all concentrations for all experiments were tested in triplicate

NUMBER OF CELLS EVALUATED: the number of revertant colonies was counted for each plate.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn; the increase in the size of the microcolonies; the reduction of the revertant colonies
- Any supplementary information relevant to cytotoxicity: any increase in the total number of revertants was evaluated for its biological relevance inclusing a comparison of the results with the historical control data range.

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.

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

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:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water, dimethyl sulfoxide and ethanol at 50 mg/ml. In THF, the test item was soluble at 100 mg/ml (= 5000 µg/plate).
- Precipitation: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 512 µg/plate and upwards.
Dose range finding test: precipitation on the plates at the end of the incubation period was also observed at the concentration of 164 µg/plate.
Mutation experiment 1: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 512 µg/plate and upwards.
Mutation experiment 2: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 492 µg/plate.
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 5% (v/v) S9-mix: 5.4, 17, 52, 164 and 512 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges, except the response for TA102 in the second mutation experiment (absence of S9-mix). Since the values were more than two-fold greater than the concurrent solvent control value, for TA102, this deviation in the mean plate count of the positive control had no effect on the validity of the results.
- Negative (solvent/vehicle) historical control data: The negative control values were within the laboratory historical control data ranges except the response for TA102 in the second mutation experiment (absence of S9-mix). Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (206 revertant colonies) when compared against relevant historical control data (225 revertant colonies), the validity of the test was considered not to be affected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies.
- Other observations when applicable: 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 TA102 (second 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 was more than two-fold greater than the concurrent solvent control value, for TA102, this deviation in the mean plate count of the positive control had no effect on the validity of the results.

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.