Lithium [ethanedioato-O,O’]tetrafluorophosphate

Genetic toxicity: in vivo

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 2013-01-09 to 2013-04-10

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Lithium [ethanedioato-O,O’]tetrafluorophosphate

EC Number:

826-676-4

Cas Number:

521065-36-1

Molecular formula:

LiPF4C2O4

IUPAC Name:

Lithium [ethanedioato-O,O’]tetrafluorophosphate

Specific details on test material used for the study:

Batch No.: 111202
Purity: 99.5%

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Young adult animals are chosen for use because of the high rate of cell division in the bone marrow, the wealth of background data on this species, and their general suitability for toxicological investigations.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: ca 40-47 days old.
- Weight at study initiation: Males weighed between 27.4 g to 32.1 g. Females weighed between 21.7 g to 25.5 g.
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Each group was kept with the sexes separated in cages and maintained in a controlled environment
- Diet: pelleted expanded rat and mouse No.1 maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 5, 10, 20, 30 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg.
- Lot/batch no.: MKBF8603V

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test substance were prepared corn oil.
Positive control solution was prepared using purified water at a concentration of 0.6 mg/mL just prior to administration

Duration of treatment / exposure:

administered on two occasions approximately 24 hours apart

Frequency of treatment:

twice

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

12.5 mg/kg bw/day (actual dose received)

Remarks:

For males

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

For males and females

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

For males and females

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

For females

No. of animals per sex per dose:

5 male/female for loe and mid dose, 7 male or 7 female for high dose group

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: oral
- Doses / concentrations: dosed once at 12 mg/kg/day

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
All the animals were killed by exposure to rising levels of carbon dioxide and both femurs dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.

DETAILS OF SLIDE PREPARATION:
The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides

METHOD OF ANALYSIS:
Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Evaluation criteria:

incidence of micronucleated polychromatic erythrocytes for the treatment group compared with the vehicle control group (p<0.05); individual and/or group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidences of micronucleated polychromatic erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent vehicle control group and where these values fall within the historical control range.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant decrease in the proportion of polychromatic erythrocytes (p<0.05).

Statistics:

For the proportion of polychromatic erythrocytes at 24 hours in male animals, an asymptotic one-tailed Jonckheere’s test for trend with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests, for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 6.
For the proportion of polychromatic erythrocytes at 24 hours in female animals, an asymptotic one-tailed Jonckheere’s test for trend with “step-down” was used on Groups 1 to 5 (excluding group 2) for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 4 (excluding group 2), then on Groups 1 and 3 (excluding group 2). Exact one-tailed Wilcoxon pairwise tests, for a decrease from control, were also carried out on Group 1 (control) versus Groups 3, 4, 5 and 6.
For incidences of micronucleated polychromatic erythrocytes at 24 hours in males, an exact one-tailed Linear-by-Linear association test with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Also, exact one-tailed pairwise Permutation tests, for an increase from control, were carried out on Group 1 (control) versus Groups 2, 3, 4 and 6.
For incidences of micronucleated polychromatic erythrocytes at 24 hours in females, an exact one-tailed Linear-by-Linear association test with “step-down” was used on Groups 1 to 5 (excluding group 2) for an increase from control. If significant, then the analysis was carried out on Groups 1 to 4 (excluding group 2). Also, exact one-tailed pairwise Permutation tests, for an increase from control, were carried out on Group 1 (control) versus Groups 3, 4, 5 and 6.
Statistical significance was declared at the 5% level for all tests.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 50-300 mg/kg/day
- Clinical signs of toxicity in test animals:
To determine suitable dose levels for use in the micronucleus test, a group consisting of two male and two female animals were administered test item at 100 mg/kg/day on two consecutive days approximately 24 hours apart.
At 100 mg/kg/day, clinical signs of toxicity were observed in one male animal only on Day 3. Clinical signs of toxicity included hindlimbs splayed, underactive behaviour, hunched posture, slow breathing and both eyelids partially closed.
On the basis of this result it was considered that the maximum tolerated dose (MTD) had not been achieved, therefore, an additional group of two male and two female animals were administered test item at 300 mg/kg/day.
At 300 mg/kg/day, clinical signs of toxicity were observed on Day 1, these included piloerection, underactive behaviour, hunched posture, slow breathing, hindlimbs splayed, reduced body temperature, salivation, prostrate posture, gasping breathing and moderate tremors. Due to the severity of the clinical signs observed the animals were killed in extremis.
On the basis of this result the MTD had been exceeded, therefore an additional group of two male and two female animals were administered test item at 200 mg/kg/day.
At 200 mg/kg/day, clinical signs of toxicity observed included underactive behaviour, hindlimbs splayed, hunched and prostrate posture, both eyelids partially closed, unsteady gait, uncoordinated gait, vocalisation, slow and irregular breathing and moderate tremors. One male animal was found dead on Day 1, the remaining male and two female animals were killed in extremis due to the severity of the clinical signs observed.
On the basis of this result the MTD had been exceeded. As a result of the inconsistent findings observed between animals previously administered test item at 100 mg/kg/day an additional group of two male and two female animals were administered test item at 100 mg/kg/day.
At 100 mg/kg/day, clinical signs of toxicity observed in male animals on Day 1 included underactive behaviour, piloerection, hunched posture, hindlimbs splayed, reduced body tone, and temperature, shallow breathing, slow breathing and urine staining. Due to the severity of the clinical signs observed both animals were killed in extremis.
At 100 mg/kg/day, clinical signs of toxicity observed in female animals on Day 1 included underactive behaviour, piloerection, both eyelids partially closed and hunched posture. The animals survived until scheduled termination with no clinical signs of toxicity displayed on Day 2 or 3.
On the basis of this result, the MTD had been exceeded for male animals. Therefore an additional group of two male animals were administered test item at 50 mg/kg/day. At 50 mg/kg/day, underactive behaviour was observed on Day 3 only.

- High dose with and without activation: the MTD in male animals was considered to be 50 mg/kg/day and the MTD in female animals was considered to be 100 mg/kg/day.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
Micronucleated polychromatic erythrocyte counts (MPCE): The test item did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male or female CD1 mice.
Micronucleated normochromatic erythrocytes (MNCE): The test item did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male or female CD1 mice.
- Proportion of polychromatic erythrocytes (%PCE): did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female CD1 mice.
- Appropriateness of dose levels and route:
The oral route was chosen for this particular study as to maximise exposure to the test system.
Animals were treated with test item at dose levels of 12.5, 25 and 50 mg/kg/day for male animals and 25, 50 and 100 mg/kg/day for female animals.No mortalities were observed during the micronucleus test. No clinical signs of toxicity were observed for the vehicle control and positive control or male animals administered test item at 12.5 mg/kg/day and female animals administrated test item at 25 and 50 mg/kg/day over the duration of the test.
At 25 mg/kg/day, one male animal displayed piloerection on Day 2 only. At 50 mg/kg/day, piloerection was observed in male animals. At 100 mg/kg/day, piloerection was observed in female animals. Some incidences of bodyweight loss were observed during the micronucleus test

Any other information on results incl. tables

-Positive Control:

Mitomycin C caused a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01) in male and female CD1 mice.

Mitomycin C did not cause a statistically significant decrease in the proportion of polychromatic erythrocytes in male or female CD1 mice.

Applicant's summary and conclusion

Conclusions:

The test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female CD1 mice when administered orally by gavage in this in vivo test procedure.

Executive summary:

The potential induction of micronuclei by test item in bone marrow cells of CD1 mice was assesses according to OECD 474. Animals were treated with test item orally by gavage on two occasions approximately 24 hours apart. A substantial difference in toxicity was observed between the sexes in the preliminary toxicity test. In line with current guidelines the micronucleus test was performed using male and female animals. In the micronucleus test male animals were administered test item at 12.5, 25 and 50 mg/kg/day, female animals were administered test item at 25, 50 and 100 mg/kg/day.

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in male or female CD1 mice treated with test item at any treatment level, compared to vehicle control values. 

It is concluded that the test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female CD1 mice when administered orally by gavage in this in vivo test procedure.