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:
30 Aug - 19 Nov 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
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): C6SFCLA
- Physical state: clear colourless liquid
- Lot/batch No.: G2X01
- Expiration date of the lot/batch: 31 Oct 2014
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 32.3 - 38.2 g
- Assigned to test groups randomly: yes, under following basis: body weight distribution
- Fasting period before study: no
- Housing: individual housing in Polycarbonate cages (TM-PC-S, Tokiwa Kagaku Kikai CO, Ltd.)
- Diet: pelleted diet for experimental animals (MF, Oriental Yeast Co., Ltd., lot No. 130612), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3-22.9
- Humidity (%): 49.9-69.2
- Air changes (per hr): 6-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil containing 10% (v/v) cremophor
- Concentration of test material in vehicle: 2.5, 5, 10 mg/L
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance was weighed, was filled up to 50% of the final volume of the vehicle and was mixed using a magnetic stirrer. The vehicle was added to the measuring glass to adjust the final volume, and mixed by gentle turning.
A 1 mL-disposable syringe with a 27 G needle was used for the intraperitoneal injection of the test substance and negative control group. A 1 mL-disposable syringe with a gastric tube was used for the oral gavage of the positive control.
Duration of treatment / exposure:
48 h (from first treatment until collection of bone marrow)
Frequency of treatment:
twice at a 24-h interval
Post exposure period:
24 h (after last treatment)
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 50, 100 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP)
- Justification for choice of positive control: commonly used positive control in micronucleus test and recommended in the applied guideline
- Route of administration: by gavage
- Doses / concentrations: 20 mg/kg bw/day
- Frequency of treatment: twice (at a 24-h interval)

Examinations

Tissues and cell types examined:
Bone marrow cell specimens
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In the dose finding study the test substance was administered intraperitoneally to 3 male and 3 female mice at 30, 100, 300, and 1000 mg/kg bw/day. All mice receiving 1000 mg/kg bw/day died after the first dosing. 2/3 male and 1/3 female of the 300 mg/kg bw/day group died after the second dosing, and a slight decrease in locomotor activity, hypothermia and tremor was observed in the surviving animals. No mortality was observed at 30 and 100 mg/kg bw/day. Therefore 25, 50, and 100 mg/kg bw/day were chosen for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treatment at 0 h and 24 h. Sampling at 48 h.

DETAILS OF SLIDE PREPARATION:
Both of the femurs were removed and the ends of the femur were cut off. Bone marrow cells were collected by washing the cavity with 0.5 mL of 10% (v/v) formalin. The cell suspension was mixed with a touch mixer, and was left to stand for 5 min to obtain the supernatant. The supernatant was mixed with 1 mL of 10% buffered formalin and centrifuged at about 170xg for 5 min. The supernatant was discarded. The precipitate was resuspended in a small amount of 10% buffered formalin, and the resulting cell suspension was stored in a serum tube at room temperature. The cell suspension was stained with the same volume of 500 µg/mL acridine orange just before microscopic observation, and was spread on a slide.

METHOD OF ANALYSIS:
Bone marrow cell specimens were observed under a fluorescence microscope with B excitation filter in a blind manner. 1000 erythrocytes per animal (500 cells per area x 2 areas of view), including both immature and mature erythrocytes, were examined to determine the percentage of immature erythrocytes (IMEs) among total erythrocytes. A total of 2000 IMEs in 2 areas of view under the microscope (1000 IMEs per area) were examined for the number of micronucleated cells (MNIMEs). Erythrocytes stained with red fluorescence in the cytoplasm were recognised as IMEs, and erythrocytes without any fluorescence in the cytoplasm were recognised as mature erythrocytes. Small bodies with yellowish green fluorescence in the cytoplasm were recognised as micronuclei. Staining was performed according the method described in Hayashi et al., An application of acridine orange fluorescent staining to the micronucleus test. Mutat. Res. 1983; 120: 241-247.
Evaluation criteria:
Criteria for a positive reaction: the test substance significantly increases the number of MNIMEs as compared to the negative control with dose-dependency.
Statistics:
Method of Kastenbaum and Bowman was applied for comparison of the number of MNIMEs in the test and negative control group. The percentage of IMEs among total erythrocytes was statistically analysed by EXSUS statistical software (Ver. 7.7.1, CAC Corporation). William´s test was applied to determine the difference between test and negative control goups regarding IMEs. F test for homogeneity of variance was applied for comparison of positive and negative controls regarding IMEs (Student´s t-test for comparison of mean values). Body weight parameters were evaluated with EXSUS software, Bartlett´s test, William´s test, Dunnett´s test, F-test, and STudent´s t-test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
100 mg/kg bw/day: reduction of ratio IME/total erythrocytes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 30 - 1000 mg/kg bw/day
- Clinical signs of toxicity in test animals: mortality and signs of toxicity observed in animals of the 300 and 1000 mg/kg bw/day dose groups
- Evidence of cytotoxicity in tissue analyzed: not performed
- Rationale for exposure: recommended in guideline
- Harvest times: not performed

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no difference between negative control and test groups
- Ratio of PCE/NCE (for Micronucleus assay): at 100 mg/kg bw/day a slight reduction (statistically significant) in the percentage of IMEs was observed, when compared to the negative control (48.4±1.9% and 54.2±2.6%)
- Appropriateness of dose levels and route: Dose levels were chosen to produce no to little toxicity according to the guideline (reduction of IMEs). No further toxicity (mortailty or clinical signs) was observed. Statistically significant differences were detected in the percentage of IMEs between the test material (100 mg/kg bw/d) and the negative controls, indicating exposure to the test material in the bone marrow.
- Statistical evaluation: appropriate statistical evaluation was performed for the various parameters

Any other information on results incl. tables

Table 1: Results from the micronucleus test in mice.

Treatment group

Dose level (mg/kg bw/day)

MNIMEs (based on 10000 IMEs scored)

IME ratio among total eryxthrocytes [%], mean of 5x1000 erythrocytes scored±SD

 

 

Number

Incidence [%], mean±SD

 

Negative control (vehicle)

0

10

0.10±0.05

54.2±2.6

Test item

25

11

0.11±0.02

53.1±1.1

Test item

50

13

0.13±0.06

53.2±2.5

Test item

100

13

0.13±0.04

48.4±1.9*

Positive control (CP)

20

71##

0.71±0.12

48.5±5.0

Historical negative control (Jan 2010-Mar 2013; 51 animals)

0

-

0.10±0.06

51.8±2.3

CP: cyclophosphamide

IME: immature erythrocyte

MNIMEs: micronucleated IMEs

##: significantly different (p≤0.01) from the negative control by Kastenbaum and Bowman´s method

*: significantly different (p≤0.05) from the negative control by William´s test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative