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 vivo

Administrative data

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

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 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

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

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day

Remarks:

Range-Finder

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day

Remarks:

Range-Finder

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Remarks:

Range-Finder

Doses / concentrations 4

Dose / conc.:

700 mg/kg bw/day

Remarks:

Range-Finder

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Range-Finder

Doses / concentrations 6

Dose / conc.:

35 mg/kg bw/day

Remarks:

Micronucleus Experiment

Doses / concentrations 7

Dose / conc.:

70 mg/kg bw/day

Remarks:

Micronucleus Experiment

Doses / concentrations 8

Dose / conc.:

175 mg/kg bw/day

Remarks:

Micronucleus Experiment

Doses / concentrations 9

Dose / conc.:

350 mg/kg bw/day

Remarks:

Micronucleus Experiment

Doses / concentrations 10

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

Examinations

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.

Results and discussion

Test results

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