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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1998
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diethyl ether
- Physical state: Liquid
- Analytical purity: 99.93%
- Lot/batch No.: 09023198
- Expiration date of the lot/batch: 09/2010
- Stability under test conditions: Not reported
- Storage condition of test material: at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann
- Age at study initiation: 6 to 12 weeks at start of acclimatisation
- Weight at study initiation: 28.5 to 33.4 g (males) and 25.3 to 29.8 g (females)
- Assigned to test groups randomly: Yes
- Fasting period before study: Not reported
- Housing: 5 animals of identical sex per cage, in IVC cage (Polysulphone), Type II L
- Diet (e.g. ad libitum): Altromin 1324 (Batch: 1130) maintenance diet for rats and mice, ad libitum.
- Water (e.g. ad libitum): Tap water, sulphur acidified to pH value of approximately 2.8 (drinking water, municipal residue control, microbiologically controlled at frequent intervals), ad libitum.
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: cotton seed oil
- Justification for choice of solvent/vehicle: The solvent was chosen according to its relative non-toxicity for the animals.
- Lot/batch no. (if required): 11KBB7604
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared and diluted in cotton seed oil within 1 hour before treatment.

Volume administered: 10 mL/kg body weight
Duration of treatment / exposure:
Single exposure with sampling of the peripheral blood being carried out at 44 hours and 68 hours after treatment.
Frequency of treatment:
Single exposure
Post exposure period:
Sampling of the peripheral blood was carried out on animals 44 h after treatment for all dose groups evaluated and 68 hours after treatment for the negative control and highest dose group (2000 mg/kg body weight).
Doses / concentrations
Remarks:
Doses / Concentrations:
400, 1000, 2000 mg/kg body weight
Basis:
other: actual injected
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide in physiological saline
- Route of administration: Intraperitoneal
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
Peripheral blood cells (at least 10000/animal)
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): The exposition times were 44 hours for all dose groups evaluated and additional 68 hours for the negative and highest dose group (2000 mg/kg body weight).


DETAILS OF SLIDE PREPARATION: Blood was obtained from tail vein after its incision. Blood cells were immediately fixed in ultracold methanol. Before analysis (at least 16 hours after fixation), fixed blood cells were washed in Hank's balanced salt solution, centrifuged at 600 x g for 5 minutes and the supernatant discarded. Blood cell populations were discriminated using specific antibodies against CD71 (expressed only at the surface of immature erythrocytes) and CD61 (expressed at the surface of platelets) and DNA content of micronuclei was determined by the use of a DNA specific strain (propidium iodide, PI).


METHOD OF ANALYSIS: Evaluation of all samples, including those of positive and negative controls, was performed using a flow cytometer. Anti-CD71 antibodies were labelled with Fluorescein-isothiocyanate (FITC), anti-CD61 antibodies were labelled with Phycoerythrin (PE). Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensity were recorded on the FL1, FL2, and FL3 channels for FITC, PE, and PI, respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test item, the ratio between immature and mature erythrocytes was determined. The results were expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).
Evaluation criteria:
There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells; and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.

According to the OECD guideline, the biological relevance as well as the statistical significance of the results are the criterion for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.
Statistics:
For the statistics, the non-parametric Mann-Whitney Test was used. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
-In the 2000 mg/kg body weight dose group, ataxia and constricted abdomen were noted in 5 males and 5 females between 2 and 30 minutes.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg body weight
- Solubility: Not reported
- Clinical signs of toxicity in test animals: Reduction of spontatneous activity, prone position, ataxia, constricted abdomen, and rough fur (piloerection)

Any other information on results incl. tables

Table 1. Relative Polychromatic Erythrocytes(PCE) (44 and 68 h preparation time)

Group

Sex

Relative PCE mean

44 h preparation time

68 h preparation time

Negative control

Males

2.12

3.31

Females

3.46

4.66

Positive control

Males

1.89

-

Females

2.83

-

2000 mg/kg

Males

2.36

3.20

Females

2.05

2.13

1000 mg/kg

Males

2.39

-

Females

3.28

-

400 mg/kg

Males

2.61

-

Females

2.25

-

No statistically significant or dose-related changes in the relative PCEs were noted.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative