Lithium bis(oxalato)borate

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 21st 1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Lithium bis(oxalato)borate

EC Number:

456-990-3

EC Name:

Lithium bis(oxalato)borate

Cas Number:

244761-29-3

Molecular formula:

C4BLiO8 (Hill Empirical Formula) C4BO8.Li (CAS Empirical Formula)

IUPAC Name:

boron(3+) lithium(1+) dioxalate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

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)

Controls

Controls 1

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

Controls 2

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).

Results and discussion

Test results

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

Applicant's summary and conclusion

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)