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Toxicological information

Genetic toxicity: in vivo

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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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Details on test material:
- Purity: 100%

Test animals

Species:
mouse
Strain:
other: Crl:CD1(ICR)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: males 31.5-31.6 g; females 24.2-24.5 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Solid bottom cages with enrichment-containing bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%):30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
3 days
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5/sex/dose at 0, 500, and 1000 mg/kg
7/sex/dose at 2000 mg/kg
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CAS 6055-19-2)
- Justification for choice of positive control(s): not reported
- Route of administration: gavage
- Doses / concentrations: 30 mg/kg

Examinations

Tissues and cell types examined:
Micronucleated reticulocytes in peripheral blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dosing concentrations used were determined by the rangefinding results from an experiment using a group of 3 male and 3 female animals. The highest dose used for the rangefinder for the in vivo micronucleus study was the limit dose of 2000 mg/kg bod weight administered by oral gavage. The animals were observed for clinical sign of toxicity and mortality immediately after dosing and daily thereafter for 3 days (until approximately 72 hours post-dosing). There were 3 test substance concentrations for the main study: a high dose, ½, and ¼ of the high-dose concentration.

TREATMENT AND SAMPLING TIMES: Based on the results from the rangefinder, each group of male and female animals was administered a single dose of the test substance at 500, 1000, or 2000 mg/kg body weight, or the vehicle or positive control substances for the main study. The vehicle and positive control groups were administered by single dose by oral gavage the same day as groups receiving test substance treatment. The test substance, vehicle, and positive controls were dosed at a volume of 10 mL/kg body weight. CP, the positive control, was administered at a concentration of 30 mg/kg body weight. Approximately 48 and 72 hours after dose administration, peripheral blood samples were collected for micronucleus evaluation. All samples were disposed of after study completion.

DETAILS OF SLIDE PREPARATION: Peripheral blood samples were collected from all animals on study. The micronucleus evaluation was conducted by flow cytometry using the In Vivo MIcroFlow® Mouse Micronucleus assay kit. Approximately 60-120 μL of blood were collected from each animal directly into a labelled microcentrifuge tube containing 350 μL anticoagulant/diluent (anticoagulant) found in the MicroFlow kit. The tubes were capped and inverted several times to mix the blood with the anticoagulant. The blood/anticoagulant mixture may have been stored at room temperature for up to 6 hours or refrigerated for up to 24 hours before fixing. The blood samples were fixed in duplicate (approximately 180 μL of blood/anticoagulant mixture each) in 2 mL ultra-cold reagent-grade methanol and stored below -75ºC until processed.

METHOD OF ANALYSIS: Evaluations were conducted on 5 animals/sex/group. At the highest concentration, 5 males and 5 females were selected for evaluation based on survival until the scheduled sacrifice and then cage order. Whenever feasible, at least 20000 RETs were analysed per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total (RETs plus NCEs). The samples were analysed and evaluated using the MicroFlow kit. The frequency of micronucleated reticulocytes (%MN-RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test substance. All groups from the 48-hour time point were analysed. For the 72-hour time point, groups 1 and 4 were analysed. Groups 2 and 3 were not evaluated since a positive response was not observed for group 4. Due to a low RET frequency and poor sample quality, the analysis of 2 animals in group 4 at the 72-hour time point were replaced with for analysis.
Evaluation criteria:
Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis. Further investigation of an equivocal result may be required to obtain a conclusive finding.

The test substance was judged negative if the following conditions were met: (1) No statistically significant dose-related increases in the group mean MN-RETs above the concurrent vehicle control value occurred at any concentration of the test substance. (2) The MN-RET values of the test substance-treated animals were within reasonable limits of the recent (past 3 years) laboratory historical control range.

The test substance was judged positive if the following conditions were met: (1) The group mean MN-RETs showed a statistically significant increase at one or more concentrations of the test substance compared to the concurrent vehicle control values. (2) An accompanying statistically significant dose-response increase in MN-RETs was observed.
Statistics:
Data for the proportion of micronucleated RETs among 20000 immature erythrocytes and the proportion of RETs among total erythrocytes (MN-RET and RET frequency, respectively) were transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. Transformed data for RET and MN-RET frequencies were analysed separately for normality of distribution and equal variance using the Shapiro-Wilk and Levene’s tests, respectively. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.

For those data that were normally distributed and had equal variance, parametric statistics (e.g., analysis of variance (ANOVA) and Dunnett’s test) were performed using the transformed data. For those data that were normally distributed but had unequal variance, a robust ANOVA and unequal-variance Dunnett test was done. For those data that were not normally distributed, nonparametric statistics (e.g., Kruskal-Wallis and Dunn’s tests) utilizing non-transformed data were performed. The individual animal was considered the experimental unit. All data analyses were one-tailed and conducted at a significance level of 5%.

No statistical analysis was conducted on body weights or clinical signs.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No statistically significant or biologically relevant increase in the micronucleated RET frequency were observed in any evaluated test substance-treated group of male or female animals at either time point. Statistically significant decreases in %RETs among the total erythrocytes were observed with both male and female animals in the 2000 mg/kg bw dose group at the 72-hour time point. These reductions indicate that the test substance reached the target cells. No other reductions in %RETs among the total erythrocytes were observed.

As expected, statistically significant increases in MNRET frequency were found in CP-treated animals of both sexes. A statistically significant depression in RET frequency was observed in male animals administered CP but not in the female animals, indicating that the 30 mg/kg bw dose level was not sufficient to induce toxicity in female animals even though it did induce MNRETs.

No clinical signs of toxicity were observed at any time point at any dose level in male or female animals exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No test substance-related mortality occurred during the study. Unscheduled mortality was observed in 3 mid-dose animals shortly after 48 hours. This mortality was caused by an unintentional technical incident at 48 hours, and not related to the test substance.

Applicant's summary and conclusion

Conclusions:
The test substance was negative in the in vivo mouse micronucleus assay.
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in peripheral blood reticulocytes (RETs) in male and female Crl:CD1(ICR) MICE. Based on rangefinding results, doses of 0, 500, 1000, and 2000 mg/kg body weight (bw) of the test substance were selected for the main study. Concurrent control groups were administered corn oil as the vehicle (negative) control, or 30 mg/kg bw of cyclophosphamide (positive control).

All animals were given a single dose by oral intubation. The vehicle control, low, intermediate, and positive control groups contained 5 animals/sex. The high-dose group contained 7 animals/sex. Peripheral blood samples were collected via tail vein bleeding, and a micronucleus evaluation was conducted by flow cytometry. Peripheral blood samples were collected approximately 48 and 72 hours post-dosing for the test substance treated and vehicle control groups, and approximately 48 hours post-dosing for the positive control group. A total of 20000 RETs were analysed per blood sample for the induction of micronuclei and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total (RETs plus normochromatic erythrocytes, NCEs). All surviving animals were weighed and observed for mortality and clinical signs of toxicity at least daily. Aliquots of the vehicle control and each test substance concentration were taken to confirm homogeneity, dose concentrations, and stability. Homogeneity and target concentrations were verified, and the test substance was stable for the duration of the dosing period.

No clinical signs of toxicity or mortality were observed in male or female animals at 2000 mg/kg bw in the range finder experiment. In the main study, no clinical signs of toxicity were observed at any time point at any dose level in male or female animals exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No test substance-related mortality occurred during the study. No statistically significant or biologically relevant increases in the micronucleated RET frequency were observed in any evaluated test substance-treated group of male or female animals at either time point. Statistically significant decreases in %RETs among the total erythrocytes were observed with both male and female animals in the 2000 mg/kg bw dose group at the 72-hour time point. These reductions indicate that the test substance reached the target cells. No other reductions in % RETs among the total erythrocytes were observed. The positive control group exhibited the expected responses resulting in statistically significant increases in mircronucleated RETs that are consistent historical control data. There were no obvious changes in body weight or body weight gain in either male or female animals administered the test substance. All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated RETs in mouse peripheral blood. The test substance was concluded to be negative in this in vivo study.