8-((3R,4S)-4-ethylpyrrolidin-3-yl)-3h-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine dihydrocloride

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:

May 3 - July 6, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride

EC Number:

845-622-0

Cas Number:

2050038-84-9

Molecular formula:

C14H19Cl2N5

IUPAC Name:

12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride

Test material form:

solid: particulate/powder

Method

Target gene:

The histidine locus in several strains of Salmonella typhimurium (Salmonella; TA98, TA100, TA1535, and TA1537) and the tryptophan locus of Escherichia coli strain WP2uvrApKM101

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor™ 1254-induced rat liver homogenate (S9) was purchased commercially and used with co-factors (S9 mix). S9 mix was prepared by standard procedures on the day of use and kept on ice until needed.

Test concentrations with justification for top dose:

The limit dose of 5000 μg/well, suggested by OECD 471, was reduced to 1000 μg/well based on the surface area of the well compared to that of a Petri plate.

The test article was evaluated in the mutagenicity assay at concentrations 0.3, 0.8, 2, 7, 22, 67, 200, 600 and 1000 μg/well.

Vehicle / solvent:

Water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Remarks:

TA98

Positive control substance:

2-nitrofluorene

benzo(a)pyrene

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Remarks:

TA100

Positive control substance:

other:

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Remarks:

TA1535

Positive control substance:

sodium azide

other:

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Remarks:

TA1537

Positive control substance:

9-aminoacridine

other:

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Remarks:

E. coli

Positive control substance:

4-nitroquinoline-N-oxide

other:

Details on test system and experimental conditions:

Tester strains were exposed to the test article via the plate incorporation methodology. In the plate incorporation methodology, the tester strain, test article, and S9 mix (where appropriate) were combined in molten supplemented top agar, which was then overlaid on minimal bottom agar in a well of a 6-well plate. Following incubation, revertant colonies were counted.

Each 6-well plate was labeled with the study number, date, test article, tester strain, activation condition, and concentrations.

Treatments in the absence of S9 were performed by adding 20 μL test or control articles, 100 μL of phosphate buffered saline (PBS) and 25 μL tester strain to sterile tubes containing 0.5 mL molten supplemented top agar (maintained at 46 ± 2°C). The mixture was overlaid onto the surface of bottom agar wells and gently spread with swirling of the plate. The plates were kept at room temperature until the top agar solidified. After the top agar solidified, the 6-well plates were inverted and incubated for two days at 37 ± 2°C. When S9 was required, 100 μL S9 mix was used instead of PBS.

The condition of the background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control. Revertant colonies were counted by hand or automated colony counter.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: These results indicate A-1335930.3 is negative (non-mutagenic) in the 6-well 5-strain bacterial reverse mutation assay under the conditions, and according to the criteria, of the study plan.

Applicant's summary and conclusion

Conclusions:

The results indicate that the test material is negative (non-mutagenic) in the 6-well 5-strain bacterial reverse mutation assay under the conditions, and according to the criteria, of the study plan.