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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh North Carolina, USA
- Age at study initiation: 8 weeks old
- Weight at study initiation: 22-31 g
- Assigned to test groups randomly: yes, computer randomization according to sex and body weight
- Fasting period before study: approximately 3 hours
- Housing: not reported
- Diet (e.g. ad libitum): ad libitum, Purina certified rodent chow 5002C
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 degrees C
- Humidity (%): 46-51%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark


IN-LIFE DATES: From: November 17, 1999 To: December 9, 1999

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: standard vehicle
- Concentration of test material in vehicle: none
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): 92706
- Purity: not reported
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 10 mL/kg


DIET PREPARATION not applicable
Duration of treatment / exposure:
single dose
Frequency of treatment:
single oral dose
Post exposure period:
2 days
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 150, 300 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5; Two additional animals per time point were dosed to account for possibility of unexpected deaths.
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C disolved in distilled water
- Justification for choice of positive control(s): standard
- Route of administration: intraperitoneal injection
- Doses / concentrations: 2.0 mg/kg

Examinations

Tissues and cell types examined:
Clinical observations: recorded on days 0 and 1 and 2 post-dosing.
- Organs examined at necropsy: : bone marrow cells from both femurs were centrifuged, spread on glass slides, air-dried, fixed in methanol, stained with Acridine Orange stain and read with an epifluorescene microscope.
For each animal a total of at least 2000 polychromatic erythrocytes was scored for the presence or absence of micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: doses were selected after evaluating the results of an acute oral toxicity study in male and femae mice


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): - Sampling times and number of samples: 24 and 48 hours post dosing. All animals from test groups and vehicle control (5/sex). Animals from the positive control groups (5/sex) at 24 hours after dosing.


DETAILS OF SLIDE PREPARATION: bone marrow cells from both femurs were centrifuged, spread on glass slides, air-dried for at least one hour, fixed in methanol fro 15 minutes and allowed to dry, stained with Acridine Orange stain and read with an epifluorescene microscope.


METHOD OF ANALYSIS: Slides from at least five animals per sex/dose group were observed when possible. Three slides were prepared per animal. Slides were coded and read blind in order to avoid bias on the part of the scorer. Slides were scanned for regios of suitable technical quality, where the cells were well spread, undamaged and well stained. These regions were normally located in a zone close to the middle of the smear. For each animal, a total of at least 1000 erythrocytes (polychromatic, referred to as PCE 1 and normochromatic) were recorded to calculate the PCE/NCE ratio. For each animal, the remaining number of polychromatic erythrocytes were recorded to total at least 2000 (referred to as PCED 2) and were scored for the presence or absence of micronuclei. Teh frequency of micronucleated polychromatic erythrocytes (MN-PCE) and the PCE/NCE ratio were calculated on the basis of these data.


OTHER: Cell counting was accomplished using the Xybion Path /Tox computer software system, G Module (GICELL program version 4.21) which captures data from the cell counter keyboard and provides an audit trail for quality assurance. Cell count data was entered, accepted and stored.
Evaluation criteria:
the test article is considered positive in this assay if it elicits a dose-response or a statistically significant increase in the number of micronucleated cells over that of the concurrent vehicle control at one or more dose levels. In the event that the test article elicits a significant increase in the number of MN-PCE due to an unusually low number of MN-PCE in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data. The test article is considered negative in this assay if: no indication of a dose-response is observed and the treatment groups do not show a statistically significant increase in the number of MN-PCE when compared to the vehicle control. - Criteria for selection of M.T.D.: the doses were selected after evaluation of the results of an acute oral toxicity in male and female CD-1 mice. The LD10 was selected as the high dose for this study since it was expected to produce significant toxicity, but not excessive lethality.
Statistics:
Data were analyzed separately for male and female animals using Statistical Analysis System (SAS, version 6.09 enhanced. An arcsine square root transformation was applied to the percent of micronucleated PCE's; all subsequent analyses for this parameter were conducted on transformed data. nitially, a three way analysis of variance model was applied to the data to determine the significance of each main effect and all two-way and three-way interaction effects. If significant interaction effects were identified, then the data were analyzed separately for each sex and/or day. Three independent single degree of freedom contrasts of the group means were used to test for trends in the group means and included in the assessment of 1) an overall effect of the test material treatment relative to control and 2) a linear dose-response trend among the tets material and 3) a quadratic dose-response trend among the test material. Additonally, pairwise comparisons between each of the three test material groups and the control group were made using Dunnett's t-test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Other: no range-finding study was conducted. Dose levels were selcted after evaluation of an acute oral toxicity study in mice.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): the test article did not induce an increase in the frequency of micronucleatedpolychromatic erythrocytes in bone marrow cells of male or female mice when compared to vehicle control values.
- Ratio of PCE/NCE (for Micronucleus assay): there was no statistically significant change in the polychromatic/normochromatic ratio at either 24 or 48 hours.
- Appropriateness of dose levels and route: Chosen after evaluating the results of an acute oral toxicity study in male and female mice
- Statistical evaluation: there was no statistically significant change in the polychromatic/normochromatic ratio at either 24 or 48 hours, which is indicative of the absence of cytotoxicity. An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, mitomycin-C. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage. Statistical methods included analysis of variance followed by Dunnett’s T-Test on Least Square Means.

Any other information on results incl. tables

MORTALITY: there were no deaths in this study. 
CLINICAL SIGNS: both male and female mice treated with 300 mg/kg of test article exhibited clinical signs of systemic
toxicity which included tremors and hyperactivity within 4 hours after treatment. Hyperactivity was also observed in
mice treated with 150 mg/kg of test article on day 0, after treatment. By day 1, all males exhibiting signs, with the
exception of one, and all females recovered.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: The test material was not mutagenic.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Under the condidtions of the study, the test material was not mutagenic in the micronucleus assay in CD-1 mouse marrow cells.
Executive summary:

The test material was evaluated for its potential to induce chromosomal damage in vivo, as assessed by the micronucleus assay with mouse bone marrow cells. Adult CD-1 male and female mice (5 male and 5 female animals per group, except for the high dose group, which had 2 additional animals per time point) received a single oral dose of the test article at concnetrations of 300, 150 or 30 mg/kg. Control animals received a single oral dose of corn oil (vehicle control), or an intraperitoneal injection of 2.0 mg/kg mitomycin-C (positve control) (MMC). Animlas from test article and vehicle control groups were euthanized at 24 or 48 hours after treatment. Animlas from the positive control group were euthanized at 24 hours after treatment. Bone marrow slides were prepared and the frequency of micronucleated polychromatic erythrocytes was measured as an indicator of cytogenetic damage. For each animal, a total of at least 2000 polychromatic erythrocytes were scored for the presence or absence of micronuclei. In addition, the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio was measured to evaluate the cytotoxicity of the test agent.

The test article did not induce an increase in the frquency of micronucleated polychromatic erythrocytes in bone marrow cells of males or female mice when compared to the vehicle controls. An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice trated with 2.0 mg/kg of the positive control, MMC. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damge.

Under the condidtions of the study, the test material was not mutagenic in the micronucleus assay in CD-1 mouse marrow cells.