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:
2006-12-21 to 2007-01-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD, USA
- Age at study initiation: Approximately 6 to 8 weeks
- Weight at study initiation:
Pilot Toxicity Study: Males: 26.0-28.7 g Females: 22.9-24.8 g
Definitive Micronucleus Study: Males: 25.5-30.1 g Females: 22.8-28.0 g
- Assigned to test groups: The mice were assigned to seven experimental groups of five males and five females each according to a computer-generated
program, which is based on distribution according to body weight.
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage.
- Diet: Ad libitum. Certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet)
- Water: Ad libitum
- Acclimation period: Not less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity: 50 ± 20%
- Air changes: Micro-VENT full ventilation, HEPA filtered system
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Corn oil
- Justification for choice of solvent/vehicle: Corn oil was chosen as the most appropriate vehicle for the test article vehicle based on test item solubility and compatibility of the vehicle with the test system. The test article was soluble in corn oil at 100 mg/mL, the maximum concentration tested in the study.
- Concentration of test material in vehicle: All dose formulations were administered by oral gavage at a dose volume of 20 mL/kg.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article dose formulations were prepared fresh for each phase of the study, just prior to dose administration. Each time, the formulations were prepared by combining an appropriate amount of test article with an appropriate amount of vehicle following by vortexing. The formulations were not adjusted for purity of the test article. All formulations appeared to be yellow solutions.
Duration of treatment / exposure:
Single oral dose
Frequency of treatment:
Single oral dose
Post exposure period:
24 h (0, 500, 1000, 2000 mg/kg bw) and 28 h (0, 2000 mg/kg bw) post-dose
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Low dose (500 mg/kg): 5 animals
Mid dose (1000 mg/kg) 5 animals
High dose (2000 mg/kg): 10 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
-Positive control: Cyclophosphamide monohydrate (CP, CAS number 6055-19-2)
- Justification for choice of positive control(s):
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg; 2.5 mg/mL

Examinations

Tissues and cell types examined:
Bone marrow was collected and bone marrow smears (slides) were prepared. Bone marrow cells (polychromatic erythrocytes) were examined microscopically for the presence of micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Selection of doses for the definitive micronucleus assay were based on the toxicity of the test article.

TREATMENT AND SAMPLING TIMES:
Groups of mice were exposed to a single oral dose at either 500, 1000 or 2000 mg/kg, or the vehicle control or the positive control article. All dose formulations were administered by oral gavage at a dose volume of 20 mL/kg. All mice in the experimental and control groups were weighed immediately before dose administration and the administered volume was based on individual body weight. Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity.

DETAILS OF SLIDE PREPARATION:
At the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation. 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. 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 resuspended by aspiration with a capillary pipette 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 fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:

Using a light microscope and a medium magnification (400X), an area of acceptable quality was selected such that the cells were well spread and stained.
Using oil immersion (1000X), the following cell populations were evaluated and enumerated:
• Polychromatic erythrocytes (PCEs)
PCEs stain bluish. Two-thousand PCEs per mouse were screened (scored) for the presence of micronuclei resulting in evaluation of a total of 10,000 PCEs per each treatment group.
• Normochromatic erythrocytes (NCEs)
NCEs stain pink (redish). 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). The incidence of MNCEs per 2000 PCEs was enumerated for each animal.
• Micronuc1ei (M)
Micronuclei are round, darkly-staining nuc1ear (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). The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes per each animal (pCEs/ECs ratio).
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10,000 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-treated animals should not be less than 20% of the control value.
All conclusions were based on a scientific judgment. As a guide to interpretation of the data, the following was considered:
• The test article was considered to induce a positive response if a dose-responsive increase in the incidence of micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p: < 0.05, Kastenbaum-Bowman Tables) at any sampling time.
• Values that were statistically significant but did not exceed the range of historical negative controls were judged as not biologically significant/relevant.
• The test article was judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution . All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Solubility Test
Corn oil was chosen as the most appropriate vehicle for the test article vehicle based on DEGDVE solubility and compatibility of the vehicle with the test system. The test article was soluble in corn oil at 100 mg/mL, the maximum concentration tested in the study.

Definitive Micronucleus Study
No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs noted within two hours after dosing included: lethargy in 3/10 high dose (2000 mg/kg) males and prostration in 1/10 high dose males. Piloerection also occurred within 2 hours of dose administration in all high dose (2000 mg/kg) animals in both sexes and again was noted in up to 3/10 high dose animals in each sex on the days following dose administration. All other mice treated with test and control articles appeared normal throughout the study.

Dose Formulation Analysis
Samples of dose formulations used in the definitive study at 0.0 mg/mL (vehicle), 25, 50 and 100 mg/mL were collected and measured.
Based on the results, concentrations of 50 and 100 mg/mL were 92.3% and 92% of target, respectively. These results indicate that the mid and highest concentrations met the acceptance criteria of ± 15 % of target and <5% RSD (relative standard deviation). The first analysis of 25 mg/mL (low concentration) failed to meet the acceptance criteria, but the repeat analysis confirmed that this concentration was within the acceptable range of target (99.4%), but the % RSD was outside of the acceptable range of <5% (6.28%). This out of target result for the low concentration did not adversely impact the outcome of the study. No test article was detected in the vehicle control samples. Overall results indicate accuracy of preparation and stability of the formulations used in this study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative