ethyl (2S,3S)-3-[(2-chloro-5-fluoro-4-pyrimidinyl)amino]bicyclo[2.2.2]octane-2-carboxylate

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:

2015-08-27 to 2015-09-17

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:

2015

Report date:

2015

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:

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

Version / remarks:

May 31, 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:

ethyl (2S)-3-[(2-chloro-5-fluoropyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2-carboxylate

EC Number:

821-183-0

Cas Number:

1777721-57-9

Molecular formula:

C15H19ClFN3O2

IUPAC Name:

ethyl (2S)-3-[(2-chloro-5-fluoropyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Physical state: solid
Appearance: White powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15AD0556
- Expiration date of the lot/batch: 2017-02-06
- Purity test date: 2015-03-16

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: The test item was not soluble in Milli-Q water and was observed to be soluble in dimethyl sulfoxide up to a concentration of 50 mg/ml. The stability of the test item in the dimethyl sulfoxide is not indicated.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in DMSO. Test item concentrations were used within 2 hours of preparation.

OTHER SPECIFICS: A correction factor of 1 for the purity/composition of the test item was applied in this study.

Method

Target gene:

The Histidine locus in 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 with and without 5% (v/v) S9-mix (TA100); based on the dose-range finding test results, a top dose of 5000 μg/plate was selected as the highest concentration in the subsequent mutation assay
Mutation experiment I: 52, 164, 512, 1600 and 5000 μg/plate at 5% (v/v) S9-mix (TA98, TA102, TA1535, TA1537)
Mutation experiment II: 492, 878, 1568, 2800 and 5000 μg/plate at 10% (v/v) S9-mix (all strains)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was not soluble in Milli-Q water and was observed to be soluble in dimethyl sulfoxide up to a concentration of 50 mg/ml. Therefore, dimethyl sulfoxide was selected as vehicle.

Controls

Controls 1

Untreated negative controls:

no

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:

no

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:

no

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:

no

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:

no

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:

no

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)
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. After this period revertant colonies were counted.

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.

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.

In addition, 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.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate decrease in the number of revertant colonies at 5000 µg/plate (Mutation experiment I); Biologically relevant decrease in the number of revertant colonies at 5000 µg/plate (Mutation experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate decrease in the number of revertant colonies at 5000 µg/plate (Mutation experiment I); Biologically relevant decrease in the number of revertant colonies at 5000 µg/plate (Mutation experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was not soluble in Milli-Q water
- Precipitation: Dose-range finding test: No precipitate was observed at the start or at the end of the incubation period; Mutation experiment I: Precipitation observed at the start (1600 and 5000 µg/plate) and the end (5000 µg/plate) of incubation; Mutation experiment II: Precipitation observed at the start (1568 µg/plate and upwards) and the end (5000 µg/plate) of incubation.

RANGE-FINDING/SCREENING STUDIES: 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. Normal bacterial background lawn and no precipitate were observed up to the highest dose. Based on the results of the dose range finding test, the dose ranges for experiment I for TA1535, TA1537, TA98 and TA102 were selected.

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
- Negative (solvent/vehicle) historical 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 in Mutation experiment II. The validity of the test is considered not to be affected, since the mean number of revertant colonies showed a characteristic number of revertant colonies (68) when compared against relevant historical control data (70).

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: Fluctuations in the number of revertant colonies below the laboratory historical control data range were observed in TA100 at 2800 µg/plate without S9 and at 1568 and 2800 µg/plate with S9, as well as fluctuations in TA102 at 1568 and 5000 µg/plate with S9 (Mutation experiment II). Since the mean number of revertant colonies is comparable to the solvent control value, these reductions are caused by incidental fluctuations in the number of revertant colonies.

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.