1-Pyrrolidinecarboxylic acid, 3-[2-[(ethoxycarbonyl)[5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl]-4-ethyl-, phenylmethyl ester, (3R,4S)-

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:

27th March 2017 to 6th June 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:

benzyl (3R,4S)-3-{2-[(ethoxycarbonyl)[5-(4-methylbenzenesulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl}-4-ethylpyrrolidine-1-carboxylate

EC Number:

944-343-2

Cas Number:

2050038-81-6

Molecular formula:

C32H35N5O7S

IUPAC Name:

benzyl (3R,4S)-3-{2-[(ethoxycarbonyl)[5-(4-methylbenzenesulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl}-4-ethylpyrrolidine-1-carboxylate

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:

N,N-Dimethylformamide (DMF)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

N,N-Dimethylformamide (DMF)

Positive controls:

yes

Remarks:

E.coli WP2uvrApKM101

Positive control substance:

4-nitroquinoline-N-oxide

other:

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

N,N-Dimethylformamide (DMF)

Positive controls:

yes

Remarks:

TA1537

Positive control substance:

9-aminoacridine

other:

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

N,N-Dimethylformamide (DMF)

Positive controls:

yes

Remarks:

TA1535

Positive control substance:

sodium azide

other: 2-aminoanthracene with metabolic action

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

N,N-Dimethylformamide (DMF)

Positive controls:

yes

Remarks:

TA100

Positive control substance:

other:

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

N,N-Dimethylformamide (DMF)

Positive controls:

yes

Remarks:

TA98

Positive control substance:

2-nitrofluorene

benzo(a)pyrene

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-1634356.0 is negative (non-mutagenic)

Applicant's summary and conclusion

Conclusions:

These results indicate A-1634356.0 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.

Executive summary:

The objective of this study was to evaluate A-1634356.0, a component of the synthetic route of ABT-494, and/or metabolites for the ability to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (Salmonella; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrApKM101 in the presence or absence of an Aroclor™ 1254-induced exogenous rat liver metabolic activation system (S9).

A-1634356.0 was evaluated in the mutagenicity assay using the plate incorporation treatment method at concentrations ranging from 0.3-1000 μg/well both with and without S9. A-1634356.0 was formulated in N,N-Dimethylformamide and formed a colorless solution at the highest concentration of 50 mg/mL. After the incubation period there was no toxicity present under either metabolic condition. Precipitate was present beginning at 200 μg/well both with and without metabolic activation. The criteria for a positive response were not met for any condition tested in the assay.

All vehicle control values were within acceptable ranges and the positive controls demonstrated the tester strains were capable of detecting mutagens. Therefore, the study was considered valid.

These results indicate A-1634356.0 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.