Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 September 1987 to 12 August 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 84-2
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): XRD-498
- Molecular formula: C12H9F2N5O2S
- Molecular weight: 326

- Physical state: tan powder
- Analytical purity: 99.8%


- Lot/batch No.: ARG 240043

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Kingston, NY
- Age at study initiation: 8 weeks

- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: single in suspended wire-bottom cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (#5002); ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 degrees C
- Humidity (%): 40-60%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil

- Concentration of test material in vehicle: 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was mixed with corn oil. Freshly prepared solutions were used for dosing the animals.

Duration of treatment / exposure:
Single oral gavage
Frequency of treatment:
Single treatment
Post exposure period:
Groups of animals were sacrificed two intervals, at 24 hours and 48 hours after treatment.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
control - corn oil - ND
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
50 mg/ml
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
167 mg/ml
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
500 mg/ml
Basis:
analytical conc.
No. of animals per sex per dose:
5 animals per sex per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Mice treated with 120 mg/kg bw cyclophosphamide were sacrificed at 24 hours, and served as positive controls.

Examinations

Tissues and cell types examined:
At the end of the specified intervals following dosing, the animals were sacrificed by cervical dislocation. Bone marrow samples were obtained from both femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A dose range finding test was conducted to aid the selection of dose levels for the micronucleus test. Groups of mice (5/sex/dose) were treated with various dose levels (5000, 2500, and 1250 mg/kg bw) of the test material. The survival of the treated mice was monitored for 5 days.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single oral gavage administered.

DETAILS OF SLIDE PREPARATION: After separting the bone from the adjoining muscles, the distal end of the femur was severed to expose the marrow cavity. A 25 gauge needle was used to aspirate the bone marrow into a 3 ml syringe containing approximately 0.5 ml of fetal bovine serum. After aspiration, the contents of the syringe were transferred into a 1.5 ml Eppendorf centrifuge tube containing 0.5 ml of serum. The cells were resuspended in the serum by gentle aspiration using the syringe and needle. The tubes were centrifuged at about 1000 rpm for approximately 5 minutes in a table-top centrifuge. The supernatant was discarded leaving a small amount of serum covering the pellet. The cell pellet was resuspended using a disposable transfer pipet. Cell smears were prepared on microscope slides using small portions of the cell suspension. Slides were allowed to air dry, fixed in methanol, and stained in 5% Giemsa.

METHOD OF ANALYSIS: Slides were coded and scored blindly. One thousand polychromatic erythrocytes were examined from each animal and the number of micronucleated polychromatic erythrocytes was recorded . Micronuclei were identified as darkly stained bodies with sharp contours and varying shapes such as round, almond, or ring. The ratio of PCE-NCE in the bone marrow was determined by examining 200 erythrocytes. The ratio was expressed as PCE x 100 / PCE + NCE.

Evaluation criteria:
Micronuclei were identified as darkly stained bodies with sharp contours and varying shapes such as round, almond, or ring. The ratio of PCE-NCE in the bone marrow was determined by examining 200 erythrocytes. The ratio was expressed as PCE x 100 / PCE + NCE. The experience of the researchers is that that ratio can be accurately estimated by counting 200 cells.
Statistics:
The raw data on the counts of MN-PCE for each animal were first transformed by adding 1 to each count and then taking natural log of the adjusted number. The transformed MN-PCE data and the data on percent PCE were analyzed by a three-way ANOVA (sex, dose, time), assuming the three-way interaction to be zero . Two way interactions were then reviewed for significance. Depending on results, data were further analyzed by either one, two, or three-way ANOVA looking only at main effects. Pairwise comparisons of treated versus negative control groups were done, if necessary, by a t-test using Bonferroni correction for multiple comparisons. The alpha level at which all the tests were conducted was 0.01.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: There were no mortalities in mice dosed with any of the dose levels (1250, 2500, and 5000 mg/kg) of the test material.

- Clinical signs of toxicity in test animals: no data
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: Since the test material is relatively non-toxic, the highest dose level for the micronucleus test was limited to 5000 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): The positive control chemical induced significant increases in the frequencies of micronucleated polychromatic erythrocytes in the bone marrows of both males and females.
- Ratio of PCE/NCE (for Micronucleus assay): The ratios of PCE to NCE observed in the groups treated with the test material were not significantly different from those of the negative control mice.
- Appropriateness of dose levels and route:
- Statistical evaluation: There were no significant differences in MN-PCE frequencies between the groups treated with test material and the negative controls. The positive control chemical induced significant increases in the frequencies of micronucleated polychromatic erythrocytes in the bone marrows of both males and females.

Any other information on results incl. tables

Table 2. Summary of the data on the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow of male mice treated with the test material or cyclophosphamide
Treatment 24 hour sacrifice 48 hour sacrifice
N MN-PCE %PCE N MN-PCE %PCE
Negative Control 5 0.2 63 5 0.2 52.2
500 mg/kg bw 5 0.2 62.6 5 0 56.6
1667 mg/kg bw 5 0 60.6 5 0 61.2
5000 mg/kg bw 5 0 64.9 5 0 64.9
CP = positive control = 120 mg/kg bw 5 31* 46.3* -- -- --
* significantly different from negative control

Table 3. Summary of the data on the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow of male mice treated with the test material or cyclophosphamide
Treatment 24 hour sacrifice 48 hour sacrifice
N MN-PCE %PCE N MN-PCE %PCE
Negative Control 5 0.4 61.4 5 0 61.5
500 mg/kg bw 5 0.2 62.3 5 0.4 68.4
1667 mg/kg bw 5 0.4 65.5 5 0.2 62.1
5000 mg/kg bw 5 0.2 67.8 5 0.4 67.5
CP = positive control = 120 mg/kg bw 5 37.6* 46.4* -- -- --
* significantly different from negative control

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It was concluded that the test material did not induce a significant increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes when given as a single oral dose to male and female CD-1 BR mice. Hence, under the experimental conditions used, the test chemical was judged negative in the mouse bone marrow micronucleus test.
Executive summary:

XRD-498 was evaluated in the mouse bone marrow micronucleus test. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. The test material was mixed with corn oil and administered to CD-1 BR mice by single oral gavage at dose levels of 0 (negative control), 500, 1667, and 5000 mg/kg body weight. Groups of animals were sacrificed at two intervals, 24 and 48 hours after treatment. Mice treated with 120 mg/kg bw cyclophosphamide and sacrificed at 24 hour served as positive controls. There were five animals per sex per dose level per sacrifice time. 1000 polychromatic erythrocytes were evalated from each animal and the frequencies of micronucleated polychromatic erythrocytes were recorded. There were no significant increase in the frequencies of MN-PCE in groups treated with the test chemical compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under the experimental conditions used, the test chemical was considered negative in the mouse bone marrow micronucleus test.