Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 October 2017 - 03 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance: pale yellow liquid
- Storage conditions: at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
These mice are recommended by international guidelines (e.g. OECD, EC)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Assigned to test groups randomly: yes
- Fasting period before study: 3-4 h before treatment until 1.5-3 h after treatment; water ad libitum.
- Weight at study initiation: 29 - 40 g
- Housing: Group housing of a maximum of 5 animals per cage in labelled Macrolon MIII cages containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO).
KG). For psychological/environmental enrichment, animals were provided with paper.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: at least 7 days

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40 - 61
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 October 2017 To: 23 February 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of vehicle: The vehicle was chosen based on stability of the test item in this vehicle
- The specific gravity of corn oil is 0.9 g/mL
Details on exposure:
JUSTIFICATION OF DOSING SOLUTIONS
The test item showed no toxicity in the dose-range finding study up to 2000 mg/kg body weight, the highest dose required in the guidelines. Therefore, 2000 mg/kg was selected as the dose to be tested.

PREPARATION OF DOSING SOLUTIONS: X-19657 was dissolved in corn oil. The X-19657 concentrations were dosed within 2 hours after preparation.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg body weight

SAMPLE COLLECTION AND ANALYSIS
Formulation samples of vehicle only and all dose concentrations groups were collected from the main study for analysis.
Formulation analysis was performed in duplicate with LC-MS/MS. Duplicate samples were analysed, the formulation was samples from the middle. and stored at -20C until analysis. Analyses were performed according to a validated method.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Twice with a 24-hour interval (at start of the experiment and after 24 hours)
Post exposure period:
Sampling of the bone marrow was done 48 hours after the first dosing.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Main study
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Main study
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Main study
No. of animals per sex per dose:
3 (DRF);
5 males (main study);
3 males (satellite groups)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: Oral, single dose
- Doses / concentrations: 40 mg/kg body weight
- Volume applied: 10 mL/kg body weight.
- Sampling: 48 hours after treatment.

Examinations

Tissues and cell types examined:
Bone marrow was collected 48 hours after the first dosing.
Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE / normochromatic erythrocytes (NCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum recommended dose is in accordance with current regulatory guidelines.

BLOOD SAMPLING AND PLASMA PREPARATION:
Blood samples were collected from 3 satellite animals of the highest dose group and of control animals per time point (1, 2, 4, 6 and 8 hours after second dosing) under iso-flurane anesthesia from the heart/vena cava ((terminal procedure). In addition, blood was collected from the animals of the vehicle and highest dose group just before bone marrow sampling. Approximately 300 μL blood samples per animal was taken and collected into tubes prepared with K2-EDTA. Within two hours after blood sampling, plasma from all samples was harvested by centrifugation for 10 minutes at 3220 g at 5°C. The plasma samples were analyzed according to a confirmed as fit for purpose bioanalytical method.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The femur bones were flushed with approximately 2 mL of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. At least two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated cover slipper.

METHOD OF ANALYSIS (Bone Marrow Smears for Micronuclei):
At first the slides were screened at a magnification of 100 x for regions of suitable technical quality.
Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).
The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

OTHER:
Mortality/Viability: at least twice a day.
Body weights: Animals were weighed individually before dosing.
Clinical signs: at least once a day.
Evaluation criteria:
ACCEPTABILITY CRITERIA
A 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 induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).

A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

EVALUATION CRITERIA
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Statistics:
ToxRat Professional v 3.2.1 was used for statistical analysis of the data.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
other: Inconclusive
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Bioanalysis showed that the bloodplasma of dosed rats did not contain the test item. This indicates that bone marrow was not exposed to the test item and therefore no conclusion on genotoxic properties of the test item can be drawn from this study.
Additional information on results:
FORMULATION ANALYSIS
The concentrations analysed in the formulations of the dose groups 200 mg/mL and 100 mg/mL were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The concentrations analysed in the formulations of 50 mg/mL were below the target concentration (mean accuracy 83.5%). No test item was detected in the solvent controls.

RESULTS OF RANGE-FINDING STUDY
No treatment related clinical signs or mortality were observed. Based on the results of the dose-range finding study, dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the micronucleus main test. Since there were no differences between sexes in toxicity only male animals were used in the main study.

RESULTS OF MAIN STUDY
- Micronucleated Polychromatic Erythrocytes
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of X-19657 treated animals compared to the vehicle treated animals.

- Ratio Polychromatic to Normochromatic Erythrocytes
The animals of the groups, which were treated with X-19657 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item on the erythropoiesis.

- Plasma concentrations
Analyzed concentrations of X-19657 in plasma samples of the vehicle control group as well as the test item treated animals were all below LLOQ (1.40 μg/mL). This indicates that the test item was not present in the blood of the animals during performance of the study and consequently that the bone marrow cells have not been exposed to the test item. Therefore no conclusion can be drawn from the study.

- Mortality and clinical signs
The negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

VALIDITY OF MAIN STUDY
- The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database.
Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

VALIDITY BIO-ANALYSIS
- QC-low, middle and high samples were analyzed in duplicate in each analytical run. Since the accuracy of ≥ 2/3 of the QC samples and ≥ 50% per concentration level fell in the criterion range of 85-115% the analytical runs were accepted.


Applicant's summary and conclusion

Conclusions:
An in vivo micronucleus test with X-19657 was performed according to OECD guideline 474 and GLP principles. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with X-19657 compared to the vehicle treated
animals. However, X-19657 was not detected in the blood plasma of dosed rats up to and including 2000 mg/kg body weight. Therefore, no conclusion can be drawn on genotoxic properties of the test item from the results obtained under the experimental conditions in this rapport.
Executive summary:

A micronucleus assay in the bone marrow of male mouse with X-19657 was performed according to OECD guideline 474 and GLP principles. Animals were dosed at the start of the experiment and after 24 hours at dose levels of 500, 1000, and 2000 mg/kg body weight. Correct dosing of the animals was confirmed by formulation analysis. No treatment related clinical signs or mortality was observed. Bone marrow was collected 48 hours after the first dosing. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of X-19657 dosed rats and the ratio of polychromatic to normochromatic erythrocytes did not decrease indicating a lack of toxic effects of X-19657 on the erythropoiesis. All acceptance criteria were met. However, bio-analysis of plasma samples indicated that the test item was not present in the blood of the animals during performance of the study indicating that bone marrow cells were not exposed to the test item. Consequently, no conclusion can be drawn on genotoxic properties of X-19657 from the results obtained in this study.