Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-Feb-2013 to 18-Feb-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH.S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use; CHMP/ICH/126642 ICH Consensus Guidelines, Step 5, December 2011.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

EC Number:

215-947-7

EC Name:

Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Cas Number:

1458-18-0

Molecular formula:

C6H5Cl2N3O2

IUPAC Name:

methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Identification: Amiloride Compound 7
Molecular formula: C6H5Cl2N3O2
Molecular weight: 222.03
CAS Number:1458-18-0
Description: Tan powder
Batch: 206-2
Purity/Composition: 99.7 area%
Test substance storage: In refrigerator (2-8°C) protected from light, desiccated
Stability under storage conditions: Stable
Expiry date: 11 May 2014 (Retest date)

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 3, 10, 33, 100, 333 and 1000 µg/plate
Experiment 2: All strains
Without and with S9-mix: 3, 10, 33, 100, 333 and 1000 µg/plate

Experiment 3: TA1535, TA98 and WP2uvrA
With S9-mix: 333, 1000 and 3330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 650 µg/plate in DMSO for TA100

Controls 2

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9; 10 µg/plate in DMSO for WP2uvrA

Controls 4

Positive control substance:

sodium azide

Remarks:

without S9; 5 µg/plate in saline for TA1535

Controls 5

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat 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 independently repeated experiment.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 3330 and 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and above in the absence of S9-mix and at 333 µg/ml in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 333 μg/plate and above in the absence of S9-mix and at 1000 µg/ml in the presence of S9-mix

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity was observed at dose levels of 333 μg/plate and above in the absence of S9-mix and at 1000 µg/ml in the presence of S9-mix

Applicant's summary and conclusion

Conclusions:

Amiloride Compound 7 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

Amiloride Compound 7 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic