Lithium bis(oxalato)borate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, Ames test, GLP, strains TA1535, TA1537, TA98, TA100, WP2uvrA, +-S9-metabolic activation, negative

OECD 476, gene mutation in mammalian cells, CHO-K1 cells, +-S9-metabolic activation, negative

OECD 473, mammalian chromosome aberration, human lymphocytes, +-S9-metabolic activation, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-08-26 to 2009-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 21st 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

May 19th 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Complete medium with 500 mL Chromosome Medium 1A with Phytohemagglutinin (Gibco) and 5 mL Penicillin/Streptomycin
Treatment medium: 500 mL Ham's F-10 and 13.1 mL Fetal calf serum
- Properly maintained: yes, in sealed tubes containing complete culture medium at 37°C

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix (Aroclor 1254 induced)

Test concentrations with justification for top dose:

0, 39.1, 78.1, 156.3, 312.5, 625 µg/mL medium

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 4 hours
- Exposure duration: 4 hours and 24 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemide
STAIN (for cytogenetic assays): Giemsa (1:10 in WEISE´s buffer, ph 6.8)

NUMBER OF REPLICATIONS: all cultures were run in duplicate using blood from a different donor.

NUMBER OF CELLS EVALUATED: For each treatment and culture 100 metaphases (about 1000 lymphocytes) were examined.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

OTHER: Haemolysis and precipitation were also examined in a preliminary toxicity test.

Evaluation criteria:

The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:

1. the number of chromosomal aberrations is significantly (at p ≤ 0.05) increased compared with the solvent control.
2. the increase observed is concentration dependent.
3. both duplicate cultures lead to similar results
4. the increase should not occur in the severely cytotoxic range (mitotic index < 0.25), as it is konwn that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests.
5. a reproducible increase in the number of cells with chromosomal aberrations.

Statistics:

The assessment was carried out by a comparison of the number of chromsome aberrations of the samples with those of the solvent control, using the exact test of R.A. Fisher (p ≤ 0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS sub committee on guidelines for mutagenic testing, part III, Statistical evaluation of mutagenicity test data, 1989).

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

was noted at the highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: no
- Precipitation: Yes, but was noted at concentrations of 1000 µg/mL and above and therefore did not effect the results of this study.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was conducted to establish the top concentration for the main cytogentic test. Seven concentrations from 10 µg/mL to 5000 µg/mL were used. In addition, one control with DMSO was tested. At the time of harvesting the concentrations from 1000 µg/mL onwards were completely cytotoxic in the experiment without S9 mix and 24 hours exposure as well as in the experiment with S9 mix and 4 hours exposure.
The solubility was assessed at the beginning and the end of treatment, as solubility could change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility would have been detected by using the unaided eye. The precipitate should not interfere with the scoring.
Each treatment was tested in the absence and presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The incidience of chromosomal aberrations (excluding gaps) of the solvent controls with and without metabolic activation and positive controls mitomycin C and cyclophosphamide were compared with control data of the last 2 years.

ADDITIONAL INFORMATION ON CYTOTOXICITY: At the time of harvesting the concentrations from 1000 µg/mL onwards in the preliminary study were completely cytotoxic in the experiment without S9 mix and 24 hours exposure as well as in the experiment with S9 mix and 4 hours exposure.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the present test conditions, lithium bis(oxalato)borate tested up to a concentration that led to test item precipitation and/or cytotoxicity an the absence and presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. In the same test, mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Executive summary:

Test samples of lithium bis(oxalato)borate were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The study was conducted according to OECD guideline 473 and EC method B.10. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate. Lithium bis(oxalato)borate was dissolved in dimethyl sulfoxide (DMSO). The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment pronounced cytotoxicity, haemolysis and test item precipitation were noted at concentrations of 1000 μg/mL and above in the experiment without and with metabolic activation (24-h or 4-h exposure). Hence, the top concentration employed in the main study was 625 μg Lithium bis(oxalato)borate/mL in the experiments without and with metabolic activation. In the main study pronounced cytotoxicity was noted at the top concentration of 625 μg Lithium bis(oxalato)borate/mL in the second experiment without metabolic activation (24 h exposure). In addition, test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 625 μg/mL medium. Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Lithium Bis(oxalato)borate at concentrations from 78.1 to 625 μg/mL (4-h exposure) or 312.5 μg/mL (24-h exposure) medium in the absence of metabolic activation ranged from 0.5 % to 2.0 %. The results obtained are considered to be within the normal range of the solvent control where a mean incidence of chromosomal aberrations (excluding gaps) of 1.0 % or 0.5 % was observed after a 4-hour and 24-hour exposure, respectively. Only at the pronounced cytotoxic concentration of 625 μg/mL medium (24-h exposure, only 45 metaphases were observed) a marginal increase (significant at p ≤ 0.05) was noted in the number of aberrations to 8.9 %. It is known that high cytotoxicity causes artefacts in form of aberrations in in vitro chromosomal tests. Hence, the increase at the concentration of 625 μg/mL medium is considered as artefact and not test item-related.

Test with metabolic activation (4-hour exposure)

The mean incidence for the 2 experiments of chromosomal aberrations (excluding gaps) of the cells treated with lithium bis(oxalato)borate at concentrations from 78.1 to 625 μg/mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5 % to 3.5 %. The results obtained are considered to be within the normal range of the solvent control where a mean incidence of chromosomal aberrations (excluding gaps) of 1.0 % or 1.5 % was observed after a 4-hour exposure, respectively.

No test item-related polyploidy or endoreduplication was noted in the experiments without or with metabolic activation. No relevant changes in pH or osmolality were noted. The range of incidence of chromosomal aberrations (excluding gaps) of the solvent controls and positive controls mitomycin C and cyclophosphamide without and with metabolic activation for experiments were compared to historical data obtained in the years 2007 - 2009. All findings with positive controls were within this range, confirming the validity of the study.

Under the present test conditions, lithium bis(oxalato)borate tested up to a concentration that led to test item precipitation and/or cytotoxicity an the absence and presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. (LPT, 2009)

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-05 to 2004-11-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Published 2000-06-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– -> trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Rat liver induced with Aroclor 1254)

Test concentrations with justification for top dose:

Combined range finding test/first mutation experiment:
TA1535, TA1537, TA98, TA100 und WP2uvrA without and with 5 % (v/v) S9-mix:
10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Second mutation assay: without and with 10 % (v/v) S9-mix:
10, 33, 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 100 without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2 uvrA without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For all tester strains with S9 mix

Details on test system and experimental conditions:

Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture ( 109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q water, and either 0.5 ml 89-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for
Escherichia coli) were counted.
The revertant colonies (histidine independent c.q. tryptophan independent) were counted manually if less than 40 colonies per plate were present. lf more than 40 colonies were present, these could be counted automatically with a Protosmodel 50000-colony counter.

Evaluation criteria:

Data evaluation and statistical procedures:

No formal hypothesis testing was done.
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagentic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test substance did not precipitate in the top agar. Precipitation of lithium bis(oxalato)borate on the plates was not observed at the start or at the end of the incubation period in all tester strains. All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related,
two-fold, increase in the number of revertants in two independently repeated experiments.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Lithium bis(oxalato)borate is not mutagenic in the Salmonella Typhimurium reverse mutation assay and the Escherichia Coli reverse mutation assay.

Executive summary:

Lithium bis(oxalato)borate was assayed for mutation in four strains of Salmonella typhimurium and one strain of Escherichia coli. according to OECD guideline 371 and EU method B.13/14. It did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies, in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Lithium bis(oxalato)borate is not mutagenic in the Salmonella Typhimurium reverse mutation assay and the Escherichia Coli reverse mutation assay. (NOTOX, 2004)

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-04 to 2011-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO cell line was originally derived from the ovary of a female Chinese hamster (Puck and Kao, 1967). The CHO KI is a sub-line of CHO cell line. The CHO KI cell line was purchased from ECACC (European Collection of Cells Cultures) (ECACC).
The cell stocks are kept in liquid nitrogen. Each batch of frozen cells was purged (GEN 013) of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office National Animal Health Institute Budapest, Hungary; results will be fully documented within the raw data file. For each experiment, one or more vials were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. When cells are growing well, subcultures were established in an appropriate number of flasks. The CHO KI cells for this study are grown in Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 and 20 hour treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants containned 10μM/mL of thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Experiment 1, 5-hour treatment period without S9 mix:
150, 400, 600, 700, 750, 800, 850*, 900* and 1000* μg/mL
Experiment 1, 5-hour treatment period with S9 mix:
300, 450, 600, 750, 800, 850, 900*, 950* and 1000* μg/mL
Experiment 2, 20-hour treatment period without S9 mix:
150, 400, 600, 700 725, 750*, 800*, 850*, 900* and 1000* μg/mL
Experiment 2, 5-hour treatment period with S9 mix:
300, 450, 600, 750, 800, 850, 900*, 950* and 1000* μg/mL
*: These concentrations were very toxic and there was not enough cells to start the phenotypic expression period after the treatment.

Vehicle / solvent:

- Vehicle used: Aqua ad injectabilia
- Justification for choice of solvent: good solubility in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Main Mutation Assay was conducted in two independent experiments in the presence and in the absence of S9 mix.
For the 5-hour treatment, 10E06 cells were placed in each of a series of sterile dishes (diameter approx. 10 mm) and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each concentration, for the untreated, negative (solvent) controls and the positive controls for treatment without and with S9 mix. On the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item. The exposure period was 5 hours. Following the exposure period the cells were washed with F12-0 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-0 medium and detached with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
In samples where sufficient cells survived, cell number was adjusted to 10E05 cells/mL. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.
Five hours treatment in the presence of S9 and 20-hour treatment in the absence of S9 mix were performed. Duplicate cultures were used for each treatment. For the treatment, 10E06 cells were placed in each of a series of sterile dishes (diameter approx. 10 mm) and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. 24 hours after the beginning of treatment the cells were washed with F12-0 medium and were detached with trypsin-EDTA solution, counted and after this, the cells were subcultured to assess cytotoxicity and to begin the phenotypic expression period.

Evaluation criteria:

The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
• The assay is valid.
• The mutant frequency at one or more doses is significantly greater than that of the relevant control.
• Increase of the mutant frequency is reproducible.
• There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) and the test item or positive control item treated groups.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- pH and osmolality:
The pH and osmolality of control and treatment solutions were measured. In osmolality no significant differences between treatment and control groups were observed in Experiments 1 and 2. As expected, the pH values decreased with increasing test substance concentrations.
- Precipitation:
Lithium bis(oxalate)borate (LiBOB) was dissolved in Aqua ad injectabilia. A clear solution was obtained up to a concentration of 20 mg/mL. For all test item concentrations examined, no precipitation in the medium was noted.

RANGE-FINDING/SCREENING STUDIES:
The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Main Mutation Assays, both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control. The results obtained were used for dose selection of the test item in the Main Mutation Assays.

COMPARISON WITH HISTORICAL CONTROL DATA:
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was very clear evidence of toxicity with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls and very clear evidence of toxicity in the absence of metabolic activation, confirming the response seen in the dose selection cytotoxicity assays. On the basis of Day 8 cloning efficiency data the cells had recovered during the expression period.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test item lithium bis(oxalate)borate (LiBOB) was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells.

Executive summary:

Lithium bis(oxalato)borate was assayed in a Mammalian Cell Gene Mutation test according to OECD guideline 476 and EU method B.17. The test item was tested in CHO-K1 cells. The test item was dissolved in Aqua ad iniectabilia and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

150, 400, 600, 700, 750, 800, 850*, 900* and 1000* μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

300, 450, 600, 750, 800, 850, 900*, 950* and 1000* μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

150, 400, 600, 700 725, 750*, 800*, 850*, 900* and 1000* μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

300, 450, 600, 750, 800, 850, 900*, 950* and 1000* μg/mL

*: These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

In Experiment 1, there were no biologically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biological differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with LiBOB in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Lithium bis(oxalate)borate (LiBOB) tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Lithium bis(oxalate)borate (LiBOB) was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells. (TOXI-COOP, 2012)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

OECD Guideline 474, in vivo micronucleus test, GLP, male mice, 250 mg/kg bw, negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015/11/27 - 2016/04/11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of GLP compliance in the study report but no certificate attached

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

These mice are recommended by international guidelines (e.g. OECD, EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 30.5 ± 1.5 g (27 - 34 g).
- Assigned to test groups randomly: yes
- Fasting period before study: 3-4 h
- Housing: The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height 180 mm, length 380 mm and width 220 mm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). A shelter (disposable paper corner home, MCORN 404, Datesand Ltd, USA) and paper bedding (Envirodri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet
- Water (e.g. ad libitum): ad libitum, tap-water
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range: 21.1 - 22.3°C)
- Humidity (%): 40 - 70% (actual range: 33 - 55%)
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: propylene glycol
- Amount of vehicle (if gavage or dermal): 5 mL/kg bw

Details on exposure:

The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Lithium-bis(oxalate) borate (LiBOB). The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use. Feed was withheld 3 - 4 hours prior to dosing until 0.5 - 2 hours after administration of Lithium-bis(oxalate) borate (LiBOB). The dosing volume was 5 ml/kg body weight. Lithium-bis(oxalate) borate (LiBOB) concentrations were prepared on the day of administration.

Frequency of treatment:

Two treatments were performed, administered at a 24-hour interval.

Post exposure period:

24 h

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral intubation
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

bone marrow erythrocyte

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study to find out the maximum tolerated dose. The test procedure and conditions were similar to those applied in the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Lithium-bis(oxalate) borate (LiBOB). Two treatments were performed, administered at a 24-hour interval. Sampling was performed 48 h after the first treatment

DETAILS OF SLIDE PREPARATION: Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, the Netherlands). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The
Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

METHOD OF ANALYSIS: To prevent bias, all slides were randomly coded before examination. An adhesive label with the study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
A test item is considered positive in the micronucleus test if:
a) At least one of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) All results are within the 95% control limits of the negative historical control data range.

Statistics:

see 'Evaluation criteria'

Key result

Sex:

male

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: 0 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: mortality at doses >= 375 mg/kg bw, at 375 mg/kg bw: ataxia; lethargy; hunched posture; rough coat

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow
- Ratio of PCE/NCE (for Micronucleus assay): The groups that were treated with 62.5 and 125 mg Lithium-bis(oxalate) borate (LiBOB)/kg body weight showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with 250 mg Lithium-bis(oxalate) borate (LiBOB)/kg body weight and cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Conclusions:

It is concluded that this test is valid and that Lithium-bis(oxalate) borate (LiBOB) is not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 250 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described. In the highest dose tested the ratio of polychromatic to monochromatic erythrocytes was significantly decreased, indicating that the test item had reached the bone marrow. Test item exposure of the target tissue was also confirmed by the analytical verification of the test substance in the serum of mice treated at the highest dose level.

Executive summary:

Cytogenicity of the test item in mice bone marrow erythrocytes was determined according to OECD Guideline 474 following GLP. As a result, Lithium-bis(oxalate) borate (LiBOB) was not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 250 mg/kg bw (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions. In the highest dose tested the ratio of polychromatic to monochromatic erythrocytes was significantly decreased, indicating that the test item had reached the bone marrow. Test item exposure of the target tissue was also confirmed by the analytical verification of the test substance in the serum of mice treated at the highest dose level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse mutation assay (Ames Test)

Lithium bis(oxalato)borate was assayed for mutation in four strains of Salmonella typhimurium and one strain of Escherichia coli. according to OECD guideline 371 and EU method B.13/14. It did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies, in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Lithium bis(oxalato)borate is not mutagenic in the Salmonella Typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay. (NOTOX, 2004)

Mammalian cell gene mutation assay

Lithium bis(oxalato)borate was assayed in a Mammalian Cell Gene Mutation test according to OECD guideline 476 and EU method B.17. The test item was tested in CHO-K1 cells. The test item was dissolved in Aqua ad iniectabilia and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

150, 400, 600, 700, 750, 800, 850*, 900* and 1000* μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

300, 450, 600, 750, 800, 850, 900*, 950* and 1000* μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

150, 400, 600, 700 725, 750*, 800*, 850*, 900* and 1000* μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

300, 450, 600, 750, 800, 850, 900*, 950* and 1000* μg/mL

*: These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

In Experiment 1, there were no biologically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biological differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with LiBOB in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Lithium bis(oxalate)borate (LiBOB) tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Lithium bis(oxalate)borate (LiBOB) was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells. (TOXICOOP, 2012)

Chromosome Aberration assay

Test samples of lithium bis(oxalato)borate were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The study was conducted according to OECD guideline 473 and EC method B.10. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate. Lithium bis(oxalato)borate was dissolved in dimethyl sulfoxide (DMSO). The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment pronounced cytotoxicity, haemolysis and test item precipitation were noted at concentrations of 1000 μg/mL and above in the experiment without and with metabolic activation (24-h or 4-h exposure). Hence, the top concentration employed in the main study was 625 μg lithium bis(oxalato)borate/mL in the experiments without and with metabolic activation. In the main study pronounced cytotoxicity was noted at the top concentration of 625 μg lithium bis(oxalato)borate/mL in the second experiment without metabolic activation (24 h exposure). In addition, test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 625 μg/mL medium. Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with lithium bis(oxalato)borate at concentrations from 78.1 to 625 μg/mL (4-h exposure) or 312.5 μg/mL (24-h exposure) medium in the absence of metabolic activation ranged from 0.5 % to 2.0 %. The results obtained are considered to be within the normal range of the solvent control where a mean incidence of chromosomal aberrations (excluding gaps) of 1.0 % or 0.5 % was observed after a 4-hour and 24-hour exposure, respectively. Only at the pronounced cytotoxic concentration of 625 μg/mL medium (24-h exposure, only 45 metaphases were observed) a marginal increase (significant at p ≤ 0.05) was noted in the number of aberrations to 8.9 %. It is known that high cytotoxicity causes artefacts in form of aberrations in in vitro chromosomal tests. Hence, the increase at the concentration of 625 μg/mL medium is considered as artefact and not test item-related.

Test with metabolic activation (4-hour exposure)

The mean incidence for the 2 experiments of chromosomal aberrations (excluding gaps) of the cells treated with lithium bis(oxalato)borate at concentrations from 78.1 to 625 μg/mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5 % to 3.5 %. The results obtained are considered to be within the normal range of the solvent control where a mean incidence of chromosomal aberrations (excluding gaps) of 1.0 % or 1.5 % was observed after a 4-hour exposure, respectively.

No test item-related polyploidy or endoreduplication was noted in the experiments without or with metabolic activation. No relevant changes in pH or osmolality were noted. The range of incidence of chromosomal aberrations (excluding gaps) of the solvent controls and positive controls mitomycin C and cyclophosphamide without and with metabolic activation for experiments were compared to historical data obtained in the years 2007 - 2009. All findings with positive controls were within this range, confirming the validity of the study.

Under the present test conditions, lithium bis(oxalato)borate tested up to a concentration that led to test item precipitation and/or cytotoxicity in the absence and presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. (LPT, 2009)

Mammalian Erythrocyte Micronucleus Test

Cytogenicity of the test item in mice bone marrow erythrocytes was determined according to OECD Guideline 474 following GLP. As a result, Lithium-bis(oxalate) borate (LiBOB) was not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 250 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions. In the highest dose tested the ratio of polychromatic to monochromatic erythrocytes was significantly decreased, indicating that the test item had reached the bone marrow. Test item exposure of the target tissue was also confirmed by the analytical verification of the test substance in the serum of mice treated at the highest dose level.

Justification for classification or non-classification

Based on the results of three in vitro and one in vivo genetic toxicity studies, lithium bis(oxalato)borate was neither found genotoxic nor mutagenic and is not subjected to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).