Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2011 - 3 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2A (1996) and ICH S2B (1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
No further details
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ-568
- Substance type: clear colorless liquid
- Physical state: liquid
- Analytical purity: 100%
- Primary amine: 97%
- Lot/batch No.: 0G704
- Storage condition of test material: at room temperature (15 to 30°C), stored in the dark without desiccant

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: dose range finding study: males and females, 7 weeks 1 or 2 days; definitive micronucleus study: males and females: 7 weeks 3 days
- Weight at study initiation: dose range finding study: male mice: 29.6 - 34.1 grams, female mice: 23.7-26.5 grams; definitive micronucleus study: male mice: 29.6-33.3 grams, female mice: 22.7-28.7 grams
- Assigned to test groups randomly: yes, under following basis: In each study, mice were assigned to the appropriate number of treatment groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ± 20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during randomization process. The cage card contained the following information: the animal number, sex, study number, test article identification, treatment group number, dose level and route of administration. Cage cards were color coded as a function of treatment group. Raw data records and specimens were also identified by the unique test animal number.

In the DRF study, mice were randomly assigned to four groups of five animals/sex, each.

In the definitive micronucleus study, mice were randomized into 8 groups (7+1) of 5 male and 5 female mice each. Animals in five of these groups were designated for 24 hour bone marrow collection, animals in the remaining two groups were designed for 48 hour bone marrow collection and animals in the 8th group were designated for the replacement in the event of mortality at the highest dose. Since mortality was not observed at the high dose, bone marrow was not collected from this group.
- Fasting period before study: no
- Housing: Animals were housed in an AAALAC-accredited facility with a controlled environment
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients
- Water (e.g. ad libitum): Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards (water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens
- Acclimation period: Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were acclimatized for 8 or 9 days in the DRF study and for 10 days in the definitive study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22.2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All formulations were administered at a dose volume of 20 mL/kg body weight with the exception of one animal in the DRF study and one animal in the definitive study that were not dosed with the appropriate volumes, as per the records in the raw data.

The test article dose formulations were prepared fresh for each phase of study prior to dose administration. All formulations for the dose range finding study (100, 50, 37.5 and 25 mg/mL) and all formulations for the definitive study (25, 12.5 and 6.25 mg/mL) were prepared as follows:
- An appropriate amount of the test article was weighed into separate containers for each concentration
- Vehicle, ~80% of the final volume was added to the respective containers.
- Each formulation was vortexed briefly and remaining volume of the vehicle was added to achieve the final volume.

All formulations appeared as clear colorless solutions.
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Single exposure
Post exposure period:
Mice in 5 groups (either positive or vehicle control articles or 125, 250 or 500 mg/kg XTJ-568) were euthanized at 24 hours post-dose. Mice in two remaining groups (either dosed with vehicle control or 500 mg/kg substance) were euthanized at 48 hours post-dose. In addition, 5 animals/sex were dosed with the substance at 500 mg/kg to be used as replacement group, in the event of mortality at the high dose. As no mortality was observed, these animals were euthanized without bone marrow collection.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate
- Justification for choice of positive control(s): no data
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A dose range finding study was performed. Five male and five female mice were exposed to XTJ-568 at 2000, 1000, 750 or 500 mg/kg. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration on Study Day 0 and on Study Days 1 and 3. Following the last observation on Study Day 3, animals were euthanized by exposure to CO2 followed by incision of the diaphragm and discarded without further examination.

DETAILS OF SLIDE PREPARATION: The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in the microscopic evaluation.

METHOD OF ANALYSIS: Scoring for micronuclei (bone marrow evaluation):
To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using a fluorescent microscope and medium magnification (400X; blue excitation filter in the rang of 440-490 nm and barrier filter combination at 520 nm), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000 X), the following cell populations and cellular components were evaluated and enumerated:
- Polychromatic erythrocytes: PCE stain orange-red. PCEs are young erythrocytes and are the target cells for evaluation of the test article genotoxicity. 2000 PCEs per each animal were screened (scored) for the presence of micronuclei (MPCEs) resulting in evaluation of a total of 10000 PCEs per each treatment group.
- Normochromatic erythrocytes: NCEs appeared light green in color. NCEs are mature erythrocytes (red blood cels) and are the final cell population formed during erythropoiesis. The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) is determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio).
- Micronuclei: Micronuclei are round, fluorescent green-stained nuclear (chromosome) fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs).
- PCEs/ECs ratio: The proportion of polychromatic erythrocytes to total erythrocytes would also be determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal. In the event of high reduction and suppression in PCEs/EC ratio (80% or greater reduction in PCEs/ECs ratio relative to the negative control), it might not be possible to evaluate 2000 PCEs per animal for the presence of micronuclei.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test article animals should not be less than 20% of the control value.
As a guide to interpretation of the data, the following was considered:
- The test article would have been considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses is statistically elevated relative to the vehicle control (p < or = 0.05, binomial distribution, Kastenbaum-Bowmantables).
- In this study, the test article was judged negative because no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the concurrent negative (vehicle groups) is observed.
However, the results of the statistical analysis would not be the only criteria in determination of the test substance potential to increase the incidence of micronucleated PCEs. A dose-dependent increase in the incidence of micronucleated PCEs or biological relevance of generated data would have been taken in consideration during determination of test article positivity. in addition, if criteria for either a positive or negative genotoxic response were not met, the results would have been judged as equivocal.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection and lethargy were observed in both male and female mice at 500 mg/kg following dose administration. Piloerection persisted in male mice.
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
All male and female mice in 2000 mg/kg treatment group were found dead following dose administration. Mortality was also observed in 1/5 male and 1/5 females at 1000 mg/kg and in 2/5 male and 1/5 female mice at 750 mg/kg. Convulsions are noted following dose administration at 1000 and 750 mg/kg. Piloerection and lethargy were among the clinical signs observed in the surviving animals at these dose levels. All mice at the dose level 500 mg/kg were convulsing immediately after dosing and exhibited piloerection and lethargy following dose administration. Piloerection persisted on Study Day 1 in some of the mice. No appreciable changes in group mean body weights were observed.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
No mortality was observed in any of the treatment groups. All mice in the control article (vehicle or positive) groups and mice in the test article groups at 125 and 250 mg/kg appeared normal during the study period. Piloerection and lethargy were observed in both male and female mice at 500 mg/kg following dose administration. Piloerection persisted in male mice.

Bone marrow evaluation:
- No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes (PCEs/ECs) in the test article groups relative to the respective vehicle control groups were observed suggesting that the test article did not inhibit erythropoiesis.
- No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test article groups relative to the respective vehicle control groups was observed in the male or female mice at 24 or 48 hours after dose administration (p > 0.05, binominal distribution, Kastenbaum-Bowman tables).
- CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p < or = 0.05, binominal distribution, Kastenbaum tables) in both male and female mice. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Any other information on results incl. tables

Deviation:

The following deviation from the protocol occurred during the conducted of the dose range finding study, which was documented in the raw data with a deviation report:

As per written records, one male mouse in the 2000 mg/kg treatment group in the DRF study was dosed with the test article at a volume of 2 mL/kg instead of 20 mL/kg. As per Study Director's opinion, the volume recorded was a documentation error since the animal was found dead, which was considered to be a result of appropriate and correct administration and exposure. Another male mouse in the vehicle control group received the vehicle at a slightly higher volume of 28.4 mL/kg.

As per Study Director's opinion, these two events did not adversely impact the dose selection for the main study or the outcome of the study.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study a single oral administration of the substance at doses up to and including 500 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the substance was concluded to be negative in the micronucleus test using male or female ICR mice.