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: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
No blood sample was collected for one animal at the 48 hour time point due to technical error. This deviation has no impact on the outcome or validity of this study.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 28 wt%

Test animals

Species:
mouse
Strain:
other: Crl:CD1(ICR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: males 30.5 g; females 24.3 - 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:
Deionized water
Details on exposure:
THe test substance, vehicle, and positive controls were administered by single oral gavage at a dose volume of 10 mL/kg body weight.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:
actual ingested
A correction factor of 3.571, based on the percent active ingredient, was used for preparation of the dosing concentrations.
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
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:
Peripheral blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dosing concentrations used were determined by the range-finding results from an experiment using groups 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 body weight administered by oral gavage. The animals were observed for clinical signs of toxicity and mortality immediately after dosing and on days 3 and 4 (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 were 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 test substance, vehicle, and positive controls were administered by single oral gavage at a dose volume of 10 mL/kg body weight. Approximately 48 and 72 hours after dose administration, peripheral blood samples were collected for all vehicle control and all test substance treated animals for micronucleus evaluation. Approximately 48 hours after dose administration, peripheral blood samples were collected from all positive control animals for micronucleus evaluation. Additional male and female animals were treated with the highest concentration to allow for unexpected mortality. All samples were disposed after study completion.

DETAILS OF SAMPLE PREPARATION: Peripheral blood samples were collected from all animals on study. Approximately 60-120 μL of blood was collected from each animal directly into a labelled microcentrifuge tube containing 350 μL anticoagulant/diluent (anticoagulant) found in the In Vivo MicroFlow® Plus Mouse Micronucleus assay kit. The tubes were capped and inverted several times to mix the blood with the anticoagulant. The blood/anticoagulant mixture were either 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 absolute methanol and stored below -75 ºC until processed.

METHOD OF ANALYSIS: The micronucleus evaluation was conducted by flow cytometry using the MicroFlow® kit. Evaluations were conducted on 5 animals/sex/group/time point. At the highest concentration, 5 males and 5 females were selected for evaluation based on survival until the scheduled sacrifice and then cage order. No blood sample was collected for one male mouse in the 1000 mg/kg dose group at the 48 hour time point due to technical error; therefore only 4 animals were analysed for the 1000 mg/kg male mice dose group at this time point. 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 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, vehicle control group and 2000 mg/kg dose group were analysed. The 500 and 1000 mg/kg dose level groups were not analysed since no positive response was observed for 2000 mg/kg dose group.
Evaluation criteria:
The individual animal was considered the experimental unit. 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 was statistically significantly increased 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.

A test was considered valid if all of the following conditions were met: (1) The range of MN-RET values in the vehicle control animals were within reasonable limits of the laboratory historical control range. (2) The positive control induced a biologically relevant increase in the frequency of MN-RETs as compared to the vehicle control group.
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.

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%.

Data from the positive control group was not included in the statistical evaluation. 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 valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
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 timepoint.
There were no statistically significant decreases in %RETs among the total erythrocytes observed in any evaluated test substance-treated group of male or female animals at either timepoint.
The positive control group exhibited the expected responses resulting in biologically relevant increases in micronucleated 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. In the main micronucleus study, clinical signs of toxicity were observed at the top dose levels. At 2000 mg/kg body weight, clinical observation of ataxia were observed in 7/7 male and 1/7 female mice approximately 1 hour after dosing. No clinical signs were observed at any other timepoint or in any other test substance treated group. No abnormalities were detected in the vehicle or positive control groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance was negative in the in vivo rat micronucleus assay.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow by analysing 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 deionized water 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. Peripheral blood samples were collected 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 dose concentrations and stability. Target concentrations were verified, and the test substance was stable for the duration of the dosing period.

In the rangefinding experiment 3 male and 3 female mice were dosed with the test substance at 2000 mg/kg bw. Shortly after dosing, clinical signs of ataxia were observed in all mice. All mice appeared to be free of clinical signs and no abnormalities were detected 48 and 72 hours after dosing.

In the main micronucleus study, clinical signs of toxicity were observed at the top dose levels. At 2000 mg/kg bw clinical observation of ataxia were observed in 7/7 male and 1/7 female mice approximately 1 hour after dosing. No clinical signs were observed at any other timepoint or in any other test substance treated group. No abnormalities were detected in the vehicle or positive control groups. 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 timepoint. There were no statistically significant decreases in %RETs among the total erythrocytes observed in any evaluated test substance-treated group of male or female animals at either timepoint. The positive control group exhibited the expected responses resulting in biologically relevant increases in micronucleated 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.