7-Cyclopentyl-N,N-dimethyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

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:

March - July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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; ICH Consensus Guideline, Step 4 of the Process, November 2011

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

7-cyclopentyl-N,N-dimethyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

EC Number:

807-111-0

Cas Number:

1211441-98-3

Molecular formula:

C23H30N8O

IUPAC Name:

7-cyclopentyl-N,N-dimethyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Test material form:

solid: bulk

Method

Target gene:

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidineindependent strains.

Species / strain

Species / strain / cell type:

other: TA1535, TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

In the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

Test concentrations with justification for top dose:

concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate
In the dose range finding test, LEE011-A4 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in tester strain TA100. LEE011-A4 precipitated only on the plates at the top dose of 5000 μg/plate in the presence of S9-mix.
Based on the results of the dose range finding test, LEE011-A4 was tested in the first mutation assay at a concentration range of 33 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA97, TA98 and TA102. LEE011-A4 did not precipitate on the plates at the top dose of 5000 μg/plate.

Vehicle / solvent:

dimethyl sulfoxide

Controls

Untreated negative controls:

yes

Remarks:

The vehicle of the test substance, which was DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: ICR-191, 2-nitrofluorene, tert-butyl hydroxide, 2-aminoanthracene

Details on test system and experimental conditions:

Salmonella typhimurium bacteria and Escherichia coli bacteria.
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA97 hisD6610/ R-factor* Frameshift
TA102 hisG428/ R-factor** Transitions/transversions
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
**: R-factor = pKM101 and pAQ1

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement,crystal violet sensitivity, ampicillin resistance (TA97, TA98, TA100 and TA102), tetracycline resistance (TA102), UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Rationale for test conditions:

The Salmonella typhimurium strains used in this study were TA1535, TA97, TA98, TA100 and TA102.
The strains TA97 and TA98 are capable of detecting frameshift mutagens; strains TA100 and TA1535 are capable of detecting base-pair substitution mutagens, and strain TA102 is capable of detecting transitions/transversions

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times (TA102) and three times (TA100, TA97, TA1537 and TA98) the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Statistics:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100, TA97 and TA102 is not greater than two (2) times the concurrent control, and the total number of
revertants in tester strains TA1535 and TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA97 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535 and TA98 is greater than three (3) times the concurrent vehicle control.
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.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Key result

Species / strain:

other: TA1535, TA97, TA98, TA100 and TA102

Remarks:

Salmonella typhimurium strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In tester strain TA98, LEE011-A4 induced up to 3.5- and 3.2-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively in the first experiment. However since these increases were not seen in the second and third experiment, no dose related response was observed and there was a large difference in the number of revertant colonies in the three replicate plates of the dose level which showed an increase in the number of revertant colonies, these increases are considered to be not biologically relevant and LEE011-A4 is considered not mutagenic.

The mean plate counts of the positive control of TA102, in the presence of S9-mix did not reach a two-fold increase compared to the concurrent vehicle control group mean.
Evaluation: Although the responses showed 1.6 to 1.7-fold increases compared to the concurrent vehicle controls, the responses were within the laboratory historical range and clear negative results were obtained in this tester strain. Therefore, this deviation in the mean plate counts of the
positive control had no effect on the results of the study.

Remarks on result:

other: not mutagenic

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that LEE011-A4 is not mutagenic in the Salmonella typhimurium reverse mutation assay.