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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES-Test (reverse mutation assay): Negative with and without metabolic activation

LEE011-A3 (=LEE011-B9) is not clastogenic or aneugenic in human lymphocytes and is also not mutagenic in the TK mutation test system.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Target gene:

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

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)

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb - July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

During or after exposure of the stimulated human lymphocytes to the test item, cells were cultured to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. Micronuclei are small particles consisting of acentric chromosome fragments (clastogenic event) or whole chromosomes (aneugenic event leading to chromosome loss), which are unable to migrate to the poles during the anaphase stage of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Peripheral human lymphocytes

Cytokinesis block (if used):

Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesisblock proliferation index (CBPI index).

Metabolic activation:

with and without

Metabolic activation system:

The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of LEE011-A3 (=LEE011-B9) for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was determined by the solubility of the test item in the culture medium.
The test item precipitated at concentrations of 63 μg/mL and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2018) are presented below:
Dose-range finding study: age 26, AGT = 12.7 h
First cytogenetic assay: age 26, AGT = 12.7 h
Second cytogenetic assay: age 27, AGT = 12.3 h

Rationale for test conditions:

Whole blood samples obtained from young healthy subjects were treated with an anticoagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic and aneugenic activity of a broad range of chemicals.

Evaluation criteria:

An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided,
p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

LEE011-A3 (=LEE011-B9) did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Conclusions:

LEE011-A3 (=LEE011-B9) is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

thymidine kinase (TK) locus in L5178Y mouse lymphoma cells,

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem mbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been
dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps. To obtain more information about the cytotoxicity, an additional test was performed with a 24 hour treatment period. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hourtreatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix.
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 250 μg/mL exposure medium in the first dose range finding test, and 125 μg/mL in the additional dose range finding test.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Rationale for test conditions:

Eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods.
Since the test item was difficult to dissolve in aqueous solutions, the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations showed a slight precipitate in the exposure medium.

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10-6).

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 106
Small and large colony mutation frequencies were calculated in an identical manner.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First Mutagenicity Test:
In the absence of S9-mix, the dose level of 63 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
In the presence of S9-mix, the dose levels of 0.49 to 16 μg/mL showed no cytotoxicity.
Therefore, the dose level of 0.49 μg/mL was not regarded relevant for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.49, 0.98, 2.0, 3.9, 7.8, 16, 31 and 125 μg/mL exposure medium.
With S9-mix: 0.98, 2.0, 3.9, 7.8, 16, 31, 63 and 125 μg/mL.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 22% compared to the total growth of the solvent controls.
In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level.

Second Mutagenicity Test
The dose levels of 0.001 to 1 μg/mL showed no cytotoxicity. Therefore, the dose levels of 0.001 and 0.01 μg/mL were not regarded relevant for mutation frequency measurement. The
dose level of 31 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were: 0.1, 0.5, 1.0, 3.9, 7.8, 16, 63 and 125 μg/mL exposure medium.
The relative total growth of the highest precipitating test item concentration was 37% compared to the total growth of the solvent controls.

Conclusions:

LEE011-A3 (=LEE011-B9) is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Evaluation of the mutagenic activity of LEE011-A3 in the salmonella thyphimurium reverse mutation assay (with independent repeat) showed that the substance is negative in a mutagenicity test.

LEE011-A3 (=LEE011-B9) did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the

historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The suspension growth over the two-day expression period for cultures treated with DMSO was between 9 and 16 (3 hour treatment) and 86 and 91 (24 hour treatment).

In the absence of S9-mix, LEE011-A3 (=LEE011-B9) did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.

In the presence of S9-mix, LEE011-A3 (=LEE011-B9) did not induce a biologically relevant increase in the mutation frequency.