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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative in genotoxicity studies in vitro and in vivo.

Link to relevant study records

Reference

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

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)

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

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 / 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

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.

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

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance is negative in genotoxicity studies in vitro and in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2013 - Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: Hayashi et al 1994; Richold et al 1990; Tweats et al 2007

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Details on species / strain selection:

young adult out-bred Han Wistar Crl:WI(Han) rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

42 male and 42 female young adult out-bred Han Wistar Crl:WI(Han) rats were obtained from Charles River (UK) Ltd., Margate, UK.
Rats not dosed in this study were transferred to Covance Laboratories Ltd. stock.
Animals were housed in wire topped, solid bottomed cages, with a maximum of three animals of the same sex per cage.
The animals were housed in rooms air-conditioned to provide 15-20 air changes/hour. The temperature and relative humidity ranges were 18 to 23C and 44 to 66%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark. The animals were routinely kept under these conditions except for short periods of time where experimental procedures dictated otherwise.
Throughout the study the animals had access ad libitum to SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd., Witham). Each batch of diet was analysed for specific constituents and contaminants.
Mains water was provided ad libitum via water bottles. The water supply was periodically analysed for specific contaminants.
Bedding was provided on a weekly basis to each cage by use of clean wood bedding (Aspen). The bedding was analysed for specific contaminants.
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.
No contaminants were expected to be present in any of the above at levels that might interfere with achieving the objective of the study. Results of any analyses performed are held centrally at Covance Laboratories Ltd.

Route of administration:

oral: gavage

Vehicle:

methylcellulose aqueous solution w/v (MC, low viscosity, M0430)

Details on exposure:

All treatments were given via oral gavage.
On arrival animals of the same sex were randomly allocated to cages. Range-Finder animals were allocated to groups of two or three and Micronucleus Experiment animals were randomised to groups of six (with the exception of positive control animals (Group 7) which were allocated to a group of three). Checks were made to ensure the weight variation of Micronucleus Experiment animals prior to dosing was minimal and did not exceed 20% of the mean weight of each sex.

Duration of treatment / exposure:

For consistency in reporting of data the following are considered equivalent: Day 1 (dosing) = 0 hours
Day 2 (dosing) = 24 hours
Day 3 (necropsy) = 48 hours.

Frequency of treatment:

The test item and vehicle control were given as two administrations, at 0 and 24 hours. This has been shown to be of sufficient duration for the expression of any genotoxic potential. A dose volume of 20 mL/kg was used for all vehicle and test item administrations, with the exception of administration of Range-Finder doses at 250, 350 and 500 and Micronucleus Experiment doses of 35 mg/kg/day, which were administered at 10 mL/kg.
The positive control was administered once only at 24 hours, with a dose volume of 10 mL/kg. All animals were sampled at 48 hours.
Individual dose volumes were based on individual body weight.

Post exposure period:

An individual record was maintained of the clinical condition of all Range-Finder Experiment and Micronucleus Experiment animals dosed in the study.
Observation times were as follows:

Post Dosing Observation Times
Animals Day Approximate observation time
Range-Finder Experiment 1 Immediate, 0.5, 1, 2 and 4-6 hours post dose
2 Pre-dose, immediate, 0.5, 1, 2 and 4-6 hours post dose
3 Equivalent times to Day 1

Micronucleus Experiment 1* Immediate, 1, 2 and 4 hours post dose
2 Pre-dose, immediate, 1, 2 and 4 hours post dose
3 Prior to necropsy
* Not required for positive control animals

Dose / conc.:

250 mg/kg bw/day

Remarks:

Range-Finder

Dose / conc.:

350 mg/kg bw/day

Remarks:

Range-Finder

Dose / conc.:

500 mg/kg bw/day

Remarks:

Range-Finder

Dose / conc.:

700 mg/kg bw/day

Remarks:

Range-Finder

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Range-Finder

Dose / conc.:

35 mg/kg bw/day

Remarks:

Micronucleus Experiment

Dose / conc.:

70 mg/kg bw/day

Remarks:

Micronucleus Experiment

Dose / conc.:

175 mg/kg bw/day

Remarks:

Micronucleus Experiment

Dose / conc.:

350 mg/kg bw/day

Remarks:

Micronucleus Experiment

Dose / conc.:

700 mg/kg bw/day

Remarks:

Micronucleus Experiment

No. of animals per sex per dose:

Dose Levels - Micronucleus Experiment

Group No. Group Description Dose level (mg/kg/day) Dose volume (mL/kg) Animal ID Sample time
1 Vehicle a 0 20 1-6M, 101-106F Day 3 (48 hours)
2 LEE011 35 10 7-12M Day 3 (48 hours)
3 LEE011 70 20 107-112F Day 3 (48 hours)
4 LEE011 175 20 13-18M Day 3 (48 hours)
5 LEE011 350 20 19-24M, 113-118F Day 3 (48 hours)
6 LEE011 700 20 119-124F Day 3 (48 hours)
7 Positive control b 20 10 25-27M, 125-127F Day 3 (48 hours)
M Male
F Female
a 0.5% methylcellulose aqueous solution w/v using purified water
b CPA administered once only at 24 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA administered once only at 24 hours
Positive Control
Dose volume (mL/kg) Concentration of CPA solution (mg/mL) Dose of CPA administered (mg/kg)
10.0 2.00 20.0

Tissues and cell types examined:

Tissue Samples
No tissues were retained from Range-Finder Experiment animals.
One femur was removed and bone marrow isolated from all Micronucleus Experiment animals at necropsy.

Details of tissue and slide preparation:

Bone Marrow Sampling and Slide Preparation
One femur from each animal was exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose (Sun et al 1999). Once the majority of the 2 mL had passed through the column a further 4 mL of serum was added to the sample tubes and loaded onto the columns.
Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, 15-25°C) and the supernatant aspirated and discarded. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again (as described above) and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. One slide per animal was immediately stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. Unstained slides were air-dried and initially stored at <-10°C with desiccant.
Following initial slide analysis it was considered necessary to perform additional slide scoring to aid data clarification. As such, the reserve slides from all animals were removed from frozen storage and stained as described above. Prior to staining (as above), the slides were re-fixed for 10 minutes in absolute methanol and rinsed several times in distilled water.
Slide Analysis
Scoring was carried out using fluorescence microscopy.
Slides from the vehicle and positive control animals were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed (blind) by an individual not connected with the dosing phase of the study.
Initially the relative proportions of PCE, seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed. Then at least 2000 PCE per animal were examined for the presence of micronuclei (MN).
In order to aid in data interpretation, the second set of slides prepared from the same animals were stained and analysed for MN PCE (2000 PCE per animal). These data were combined with the initial set of data with discussion and interpretation based on these combined animal scores.
Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories standard operating procedures. The analysts were physically located remote from the Covance facility, but were subject to Covance management and GLP control systems (including QA inspection). All slides and raw data generated by the remote analysts have been returned to Covance Laboratories for archiving on completion of analysis.

Evaluation criteria:

For valid data, the test item was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. The group mean MN PCE value at such a point exceeded the 95% calculated confidence interval for the mean historical vehicle control data
4. A dose-response trend in the proportion of MN PCE was observed.
The test item was considered positive in this assay if all of the above criteria were met. The test item was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al 1990). Biological relevance was taken into account, for example consistency of response within and between dose levels.

Statistics:

After completion of microscopic analysis and decoding of the data the following were calculated:
1. %PCE for each animal and the mean for each group. The group mean %PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per 2000 PCE) and %MN PCE for each animal and the group mean %MN PCE (± standard deviation).
The MN PCE data from the vehicle control group(s) were compared with the laboratory's historical vehicle control ranges (individual animal distribution data and calculated group mean 95% confidence interval) to determine whether the assay was acceptable. For each group, inter-individual variation in the numbers of MN PCE was estimated initially by means of a heterogeneity chi-square calculation (Lovell et al 1989). For the female data set, the heterogeneity test indicated statistical significance such that analysis of the data via use of ranks was considered more appropriate. As such the numbers of MN PCE in each treated female group were compared with the numbers in vehicle control groups by use of the Wilcoxon rank sum test (Lehmann 1975). For males, the numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square (Lovell et al 1989).
For both sets of data, the tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

up to 350 mg/kg/day

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The substance is negative in genotoxicity studies in vitro and in vivo.

It is concluded that LEE011 did not induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of male rats treated up to 350 mg/kg/day (an estimate of the maximum tolerated dose) under the experimental conditions employed. In females small equivocal increases in MN were observed but were considered of highly questionable biological relevance.