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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2015-03-04 to 2015-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
no
Remarks:
non GLP, but in compliance with China National Metrology Accreditation
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Lithium bis[ethanedioato(2-)-κO1,κO2]difluorophosphate(1-)
EC Number:
695-938-6
Cas Number:
678966-16-0
Molecular formula:
F2C4PLiO8
IUPAC Name:
Lithium bis[ethanedioato(2-)-κO1,κO2]difluorophosphate(1-)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch No.: 141104
Purity: 95.4%

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Zhejiang Center of Laboratory Animals, Hangzhou, Zhejiang
- Weight at study initiation: 25 - 30 g
- Assigned to test groups randomly: Yes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 °C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light /dark cycle
- Air changes (per hr): 10 - 12

Administration / exposure

Route of administration:
other: negative control group and the nominal treatmeht groups: gavage; positive controI group: intraperitoneaI injection
Vehicle:
- Vehicle used: Vegetable oil
- Concentration of test material in vehicle: 25, 50, 100 mg/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance in vegetable oil were prepared
Duration of treatment / exposure:
All mice were administered again 24h after the first dosing.
Frequency of treatment:
twice, with an interval 24 h
Doses / concentrationsopen allclose all
Dose / conc.:
25 other: mg/kg
Dose / conc.:
50 other: mg/kg
Dose / conc.:
100 other: mg/kg
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 40 mg/kg (dissolved in physiologic solution)

Examinations

Tissues and cell types examined:
Bone marrow from sternum
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
For the micronucleus assay, sternum was removed and marrow was aspirated from the bone and transferred to glass slides containing a drop of fetal calf serum. After the slides were allowed to air-dry, they were fixed in absolute methanol for 15 min, air-dried again. The bone marrow samples were stained with Giemsa solution. Briefly, Giemsa was prepared in a 1:9 solution with phosphate-buffered saline (PH 6.8), and slides were stained for approximately 20 min. The slides were then rinsed with phosphate-buffered saline (PH 6.8).

METHOD OF ANALYSIS:
Under a microscope, at least 2000 polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MNPCE) per 200 cells was calculated, and the ratio PCE/NCE was determined by counting a total of 200 erythrocytes. PCEs with more than one micronucleus were scored as a single MNPCE. The frequency of micronuclei in polychromatic cells provided an index of induced genetic damage.
Statistics:
The frequency of micronucleated PCE (MNPCE/200) scores was transformed by the Square Root Transformation approach, and the normality of the distribution of the transformed date was assessed by means of the method of moments. The control and treated groups were then compared by using the Dunnett’s – test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clinical Signs / Mortality:
No clinical signs were observed in any test animal which could be suspected to be due to the test substance. And no death occurred in any test animal.

Micronucleus rate/ PCE/NCE ratio:
A statistically significant increase in the incidence of MNPCE and decrease in the polychromatic erythrocyte fraction over the control value was observed following treatment with the positive control substance cyclophosphamide ( P < 0.01), relative to the negative control in either male or female mouse bone marrow. The values for three treated doses for males and females did not reach statistical significance (p>0.05) when the treatment and negative control groups were compared.

Applicant's summary and conclusion

Conclusions:
No effects on the induction of MNPCE were observed with the test agent in three different doses for males and females.
Executive summary:

The erythrocyte micronucleus test of the test substance in male and female ICR mice has been studied according to OECD Guideline 474.

25 male and 25 female ICR mice were randomly assigned to 5 groups. The positive controls were treated with cyclophosphamide at the concentration of 40 mg/kg and the negative controls received excipient only. The male and female sample groups were dosed with the test substance at doses of 25, 50 and 100 mg/kg twice, with an interval 24 h. Bone marrow was harvested 20h after the last dosing and analyzed for frequency of MNPCE. A minimum of 2000 polychromatic erythrocytes (PCEs) was analyzed for each animal. The ratio PCE / NCE was determined by counting a minimum of 200 erythrocytes (PCEs plus normachromatic erythrocytes [NCEs] ).

 

No clinical signs were observed in any test animal which could be suspected to be due to the test substance. And no death occurred in any test animal.

A statistically significant increase in the incidence of MNPCE and decrease in the polychromatic erythrocyte fraction over the control value was observed following treatment with the positive control substance cyclophosphamide ( P < 0.01), relative to the negative control in either male or female mouse bone marrow. The values for three treated doses for males and females did not reach statistical significance (p > 0.05) when the treatment and negative control groups were compared.

 

Results indicated that no effects on the induction of MNPCE were observed with the test agent in three different doses for males and females.

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