Copper(2+) tetrafluoroborate(1-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Potassium tetrafluoroborate

EC Number:

237-928-2

EC Name:

Potassium tetrafluoroborate

Cas Number:

14075-53-7

IUPAC Name:

potassium tetrafluoroborate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Potassium tetrafluoroborate
- Physical state: white powder, crystalline, odourless
- Analytical purity: 99.1%
- Lot/batch No.: BWF10626
- Expiration date of the lot/batch: 31 July 2013
- Storage condition of test material: ambient temperature

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

2.5, 5.0, 10, 39, 79, 158, 315, 630, 1260 μg/mL (first assay)
125, 250, 500, 650, 800, 1000, 1260 µg/mL (second assay)

Vehicle / solvent:

- Solvent used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: vinblastine sulphate

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

NUMBER OF REPLICATIONS:
Two independent in vitro micronucleus tests were conducted for which blood was obtained from two different donors.

DURATION
- Preincubation period: 48 hours
- Exposure/Recovery: In a first test, in the presence and absence of metabolic activation (S9-mix) the treatment/recovery time was 4/20 hours (pulse treatment). In a second test, in the absence of metabolic activation, the treatment/recovery time was 20/28 hours (continuous treatment group).

NUMBER OF CELLS EVALUATED:
2000 binucleated cells per concentration (1000 per culture) were examined for the presence of micronuclei.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). The CBPI indicates the average number of cell cycles per cell during the period of exposure to Cytochalasin B, and was used to calculate cell proliferation. The CBPI was determined from at least 500 cells per slide (in total 1000 cells per dose level) and was used to estimate the percentage of cytotoxicity by comparing values in the treated and control cultures.
METHOD OF APPLICATION: in medium

DURATION
First assay
- Preincubation period: 48 hours
- Exposure duration: 4 hours (pulse treatment)
- Recovery time (cells in growth medium): 20 hours
- Harvest time (start of exposure up to harvest of cells): 24 hours
Second assay
- Preincubation period: 48 hours
- Exposure duration: 20 hours (continuous treatment)
- Recovery time (cells in growth medium): 28 hours
- Harvest time (start of exposure up to harvest of cells): 48 hours

STAIN (for cytogenetic assays): acridin-orange

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000 cells per slide were examined for the presence of micronuclei, 500 cells per slide were used to determine cytotoxicity

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation index (CBPI)

Evaluation criteria:

The study was considered valid if the selected clastogenic and aneugenic positive controls gave a statistically significantly increase in the number of binucleated cells containing micronuclei and the negative controls were within the historical data presented by test facility.

A response was considered positive if a statistically significant concentration-related or a reproducible statistically significant increase in the number of binucleated cells containing micronuclei was induced, at any of the test points.

A response was considered equivocal if the percentage of binucleated cells containing micronuclei was statistically marginal higher than that of the negative control (0.05
A test substance was considered negative if it produces neither a statistically significant concentration-related or reproducible statistically significant increase in the number of binucleated cells containing micronuclei, at any of the test points.

Statistics:

The frequencies of micronuclei found in the cultures treated with the test substance and positive control cultures were compared with those of the concurrent solvent control using Fisher's exact probability test (one-sided). The results were considered statistically significant when the p-value of the Fisher’s exact probability test was less than 0.05 (P<0.05).

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first assay, analysis of micronuclei formation was carried out at three dose levels (1260, 630 and 315 μg/mL), in the cultures of the solvent control and in the cultures of the positive controls. In the second assay, analysis of micronuclei formation was carried out in the cultures of three dose levels (1260, 1000 and 800 μg/mL) of the test substance, the cultures of the solvent control and the cultures of the positive controls.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the first and second in vitro micronucleus test, the numbers of binucleated cells containing a micronucleus in the negative control cultures were compared to the historical data. In both first and second in vitro micronucleus test, the negative controls were comparable with the the historical data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

In a GLP-compliant micronucleus test, performed according to OECD Guideline 487, potassium tetrafluoroborate was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9 -mix). Two independent in vitro micronucleus tests were conducted for which blood was obtained from two different donors. Dimethylsulfoxide (DMSO) was used as solvent for the test substance. Dose levels ranging from 2.5 to 1260 μg/mL were tested as final concentrations in the culture medium. Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment/recovery time was 4/20 hours (pulse treatment). In the second test, in the absence of metabolic activation, the treatment/recovery time was 20/28 hours (continuous treatment group). In the first test, in the presence of metabolic activation, a trend towards slightly decreased CBPI-values was observed at all dose levels. In the absence of metabolic activation, no cytotoxicity was observed. In the pulse treatment test, analysis of micronuclei formation was carried out at three dose levels (1260, 630 and 315 μg/mL), in the cultures of the solvent control and in the cultures of the positive controls. In the second test, no cytotoxicity was observed at any of the dose levels analysed. In the continuous treatment group, analysis of micronuclei formation was carried out in the cultures of three dose levels (1260, 1000 and 800 μg/mL) of the test substance, the cultures of the solvent control and the cultures of the positive controls. In both the first and the second test, the negative controls were within the historical data. Treatment with the positive controls, cyclophosphamide, vinblastine sulphate and mitomycin C, resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the cultures treated with the solvent control. This demonstrates the validity of the study. In both the first and second test, the test substance did not cause a significant increase in the number of binucleated cells containing micronuclei, at any of the dose levels analysed, when compared to the numbers found in the concurrent negative control. From the results obtained in the first and second in vitro micronucleus test it is concluded that, under the conditions used in this study, the test substance potassium tetrafluoroborate was not clastogenic and/or aneugenic to cultured human lymphocytes.