Lithium [ethanedioato-O,O’]tetrafluorophosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-05-01 to 2012-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration 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%

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: Human lymphocytes have been used in this type of study for a number of years.
- Normal cell cycle time (negative control): 14 h

For lymphocytes:
- Sex, age and number of blood donors: two healthy, non-smoking male donors
- Whether whole blood or separated lymphocytes were used: whole blood are used
- Whether blood from different donors were pooled or not: yes
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 fraction was obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver. The S9 fraction was purchased from a commercial source and stored at -80°C or below.
- method of preparation of S9 mix: S9 mix contained: S9 fraction (10% v/v : test 1 and 25% v/v : test 2), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of S9 mix (2% v/v final concentration)

Test concentrations with justification for top dose:

- First test: 20.35, 33.92, 56.52, 94.21, 157.02, 261.70, 436.17, 726.95, 1211.58 and 2019.3 μg/mL with or without S9 mix
- Second test:
In the absence of S9 mix: 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 and 700 μg/mL.
In the presence of S9 mix: 400, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400 and 1500 μg/mL.

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item was soluble in DMSO at 201.93 mg/mL (1M). On dosing a 201.93 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 2019.3 μg/mL (10 mM), no precipitate was observed.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added to cultures at 1% v/v (50 μL per 5 mL culture).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: approximately 48 hours
- Exposure duration/duration of treatment: First test:3 h; Second test: 21 h without S9 mix, 3h with S9 mix
- Harvest time after the end of treatment (sampling/recovery times): 18 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at
500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4°C until slide analysis was completed.
- Microscopic examination: The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity. From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the vehicle control value was the highest concentration selected for metaphase analysis.
Intermediate and low concentrations were also selected.
Where no significant decrease in toxicity was observed (i.e. no significant reduction in mitotic index greater than 50%), the maximum concentration tested was the highest concentration to permit metaphase analysis. Lower concentrations were also selected.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred metaphase figures were examined from each culture. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.
As a result of the observed statistically increase in polyploidy and endoreduplicated cells, the incidence of polyploid and endoreduplicated cells, out of 500 metaphase cells, was also determined quantitatively for all cultures used for chromosomal analysis under the same treatment condition.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic indices

Statistics:

The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

FIRST TEST
- Toxicity data:
In the absence of S9 mix following 3 hour treatment, test item caused a reduction in the mitotic index to 55% of the vehicle control value at 726.95 μg/mL. The concentrations selected for metaphase analysis were 94.21, 436.17 and 726.95 μg/mL.
In the presence of S9 mix (2% v/v final concentration) following 3 hour treatment, test item caused a reduction in the mitotic index to 74% of the vehicle control value at 1211.58 g/mL. The concentrations selected for metaphase analysis were 157.02, 436.17 and 1211.58 μg/mL.
- Genotoxicity results:
In the absence of S9 mix (3 hour treatment):
test item caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared to the vehicle control.
Statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis at concentrations of 436.17 μg/mL (p<0.01) and 726.95 μg/mL (p<0.001), when compared to the vehicle control. No statistically significant increases were observed at the lowest analysed concentration of 94.21 μg/mL, when compared to the vehicle control. As a result of the observed statistically significant increases in polyploidy, the incidence of polyploid metaphase cells, out of 500 metaphase cells (where possible), was also determined quantitatively for all cultures in used in chromosomal analysis under the same treatment condition.
The positive control (Mitomycin C) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system.
In the presence of S9 mix (3 hour treatment):
test item caused a statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at 1211.58 μg/mL (including gaps only), when compared to the vehicle control. However, as the observed increase was within the laboratory historical control range, when taken at the 99% confidence limit, this increase was considered to have been exaggerated by the low vehicle control values, and therefore considered biologically non-relevant. No statistically significant increases were observed at the lower analysed concentrations of 157.02 μg/mL or 436.17 μg/mL, when compared to the vehicle control.
A statistically significant increase in the proportion of polyploidy cells were observed during metaphase analysis at 1211.58 μg/mL (p<0.001), when compared to the vehicle control. No statistically significant increases were observed at the lower analysed concentrations of 157.02 μg/mL and 436.17 μg/mL, when compared to the vehicle control.
A statistically significant increase in the proportion of endoreduplicated cells was observed during metaphase analysis at 1211.58 μg/mL only (p<0.001), when compared to the vehicle control. No statistically significant increases were observed at the lower tested concentrations of 157.02 μg/mL and 436.17 μg/mL, when compared to the vehicle control.
As a result of the observed statistical increases in polyploidy and endoreduplication, the incidence of polyploid and endoreduplicated cells, out of 500 metaphase cells (where possible), was also determined quantitatively for all cultures in used in chromosomal analysis under the same treatment condition.
The positive control (Cyclophosphamide) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

SECOND TEST
- Toxicity data:
In the absence of S9 mix following 21 hour continuous treatment, test item caused a reduction in the mitotic index to 48% of the vehicle control value at 150 μg/mL. The concentrations selected for metaphase analysis were 25, 75 and 150 μg/mL.
In the presence of S9 mix (5% v/v final concentration) following 3 hour treatment, test item caused a reduction in the mitotic index to 54% of the vehicle control value at 1500 μg/mL. The concentrations selected for metaphase analysis were 1100, 1300 and 1500 μg/mL.
- Genotoxicity results:
In the absence of S9 mix (21 hour continuous treatment):
test item caused statistically significant increases (p<0.001: including and excluding gaps) in the proportion of cells with chromosomal aberrations at 150 μg/mL only, when compared to the vehicle control. No statistically significant increases were observed at the lower analysed concentrations of 25 μg/mL or 75 μg/mL, when compared to the vehicle control. All mean values for the vehicle control (DMSO) and the 25 μg/mL and 75 μg/mL test item treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit. However, at 150 μg/mL, the highest analysed concentration, statistically significant increases (p<0.001) were observed in the proportion of aberrant cells, which clearly exceeded the laboratory historical control range. No statistically significant increases in the proportion of polyploid or endoreduplicated cells were observed during metaphase analysis at any concentration, when compared to the vehicle control.
The positive control (Mitomycin C) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system.
In the presence of S9 mix (3 hour treatment):
test item caused statistically significant increases (p<0.001: including and excluding gaps) in the proportion of cells with chromosomal aberrations at all analysed concentrations (1100, 1300 and 1500 μg/mL), when compared to the vehicle control. All mean values for the vehicle control (DMSO) were within the laboratory historical control range, when taken at the 99% confidence limit. All test item treatment concentration analysed clearly exceeded the laboratory historical control range.
Statistically significant increases in the proportion of polyploidy cells were observed during metaphase analysis at 1100 (p<0.001), 1300 μg/mL (p<0.001) and 1500 μg/mL (p<0.01), when compared to the vehicle control. A statistically significant increase in the proportion of endoreduplicated cells was observed during metaphase analysis at 1100 μg/mL (p<0.001) and 1300 μg/mL (p<0.01), when compared to the vehicle control.
As a result of the observed statistical increase in the incidence of polyploidy and endoreduplicated cells during metaphase analysis, the incidence of polyploid and endoreduplicated cells, out of 500 metaphase cells (where possible), was also determined quantitatively for all cultures in used in chromosomal analysis under the same treatment condition.
The positive control (Cyclophosphamide) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item has shown evidence of causing an increase in the frequency of structural chromosome aberrations at concentrations of 150 μg/mL, in the in the absence of S9 mix following continuous treatment, and at all test item treatment concentrations (1100, 1300 and 1500 μg/mL) in the presence of S9 mix (5% v/v), in this in vitro cytogenetic test system under the conditions described. In addition, the test item has shown significant increases in numerical aberrations in the form of polyploidy, and morphological changes in the form of endoreduplication, in this in vitro cytogenetic test system.

Executive summary:

The ability of test item to induce chromosomal aberrations in human lymphocytes cultured in vitro was assessed based on OECD 473.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to LiPF4(Ox) both in the absence and presence of S9 mix derived from rat livers. Vehicle and positive control cultures were also included.

The study comprised of two independent tests. In the first test, a 3 hour treatment was used in both the absence and presence of S9 mix. In the second test, a 21 hour continuous treatment was used in the absence of S9 mix, and a 3 hour treatment using an increased S9 concentration (5% v/v) was used in the presence of S9 mix

The test concentrations are listed as follows:

First test:20.35, 33.92, 56.52, 94.21, 157.02, 261.70, 436.17, 726.95, 1211.58 and 2019.3 μg/mL with or without S9 mix

Second test: Without S9 mix: 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 and 700 μg/mL; with S9 mix: 400, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400 and 1500 μg/mL.

The test item has shown evidence of causing an increase in the frequency of structural chromosome aberrations at concentrations of 150 μg/mL, in the in the absence of S9 mix following continuous treatment, and at all test item treatment concentrations (1100, 1300 and 1500 μg/mL) in the presence of S9 mix (5% v/v), in this in vitro cytogenetic test system under the conditions described. In addition, test item has shown significant increases in numerical aberrations in the form of polyploidy, and morphological changes in the form of endoreduplication, in this in vitro cytogenetic test system.