tert-Butyl 4-({6-[7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}pyridin-3-yl)piperazine-1-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:

Feb - May 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:

2018

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)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-Butyl 4-({6-[7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}pyridin-3-yl)piperazine-1-carboxylate

EC Number:

810-753-4

Cas Number:

1374639-78-7

Molecular formula:

C28H38N8O3

IUPAC Name:

tert-Butyl 4-({6-[7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}pyridin-3-yl)piperazine-1-carboxylate

Test material form:

solid: bulk

Method

Target gene:

reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidineindependent strains.

Species / strain

Species / strain / cell type:

S. typhimurium, other: Escherichia coli

Remarks:

TA1535, TA97, TA98, TA100 and TA102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with strain TA100 with and without 5% (v/v) S9-mix.
Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate were tested in triplicate. Due to precipitate of the test substance on the plates, the highest concentration of LEE011-A3 used in the subsequent mutation assay was 333 μg/plate.

Vehicle / solvent:

dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany)

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

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).
Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 36.3 – 39.0°C) in the dark. Temporary deviations of maximally 2 hours (in the range of 38.0 – 39.0°C) occurred due to addition of plates (which were at room temperature) to the incubator or
due to opening and closing the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity

Rationale for test conditions:

The Salmonella typhimurium reverse mutation assay has been shown to be rapid and adequate indicators for the mutagenic activity of a wide range of chemical compounds.
The assay was conducted in the absence and presence of a metabolizing system (S9-mix).
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:

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:

S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The solvent control of tester strain TA97 (second experiment, absence of S9-mix) showed a response (mean plate count) which was not within the laboratory historical range.
Evaluation: The value (175) was above the limit of the range (169). The mean plate count was just outside the limit of the range and clear negative results are observed in all experiments, therefore this deviation in the mean plate count of the solvent control had no effect on the results of the study.
2. The mean plate counts of the positive control substance of tester strain TA102 did not reach a two-fold increase compared to the concurrent vehicle control group mean in some experiments.
Evaluation: Although the response of the positive control substance in the second experiment was just outside the laboratory historical range, the responses showed 1.5 and 1.8-fold increases compared to the concurrent vehicle controls 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 The study integrity was not adversely affected by the deviations.

Any other information on results incl. tables

LEE011-A3 was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA98, TA102 and TA97 in the absence and presence of S9-mix: 3, 10, 33, 100 and 333 μg/plate.

Precipitate

No precipitation of LEE011-A3 on the plates was observed at the start of the incubation period however at the end of the incubation period precipitate was observed at the concentration of 333 μg/plate.

Toxicity

There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity

No increase in the number of revertants was observed upon treatment with LEE011-A3 under all conditions tested.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant doserelated increase in the number of revertants in two independently repeated experiments.

Based on the results of this study it is concluded that LEE011-A3 is not mutagenic in the Salmonella typhimurium reverse mutation assay.