Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Since all the available in vitro tests showed negative results, potassium tetrafluoroborate is not to be considered as mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, published in peer-reviewed literature, minor restrictions in reporting, but otherwise acceptable for assessment.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
polychlorinated biphenyl induced rat liver S9-mix
Test concentrations with justification for top dose:
1, 5, 10, 50, 100, 500, 1000 and 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:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Remarks:
strains TA 100, TA 98 and WP 2 uvrA, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
strain 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:
strain TA 1537, 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:
strain TA 1538, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
strains TA 100, TA 98, TA 1537 and TA 1538, with 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:
strains TA 1535 and WP 2 uvrA, with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Incubation period: 48 hours

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: background lawn
Species / strain:
other: TA98, TA100, TA1535, TA1537 and TA1538
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
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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
Positive controls validity:
valid
Conclusions:
The test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

An Ames test with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli strain WP2uvrA was performed with tetrafluoroboric acid (42% aqueous solution). The study was conducted according to the Guidelines of the Japanese Ministry of Labour. The strains were treated with test concentrations ranging from 1 to 5000 μg/plate, both in the presence and absence of metabolic activation. No increased number of revertants was observed in any case. Based on the results, the substance was concluded to give negative results in the Ames test.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
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.
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
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).
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.
Conclusions:
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
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.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus on chromosome 11.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
0.62, 1.2, 2.5, 4.9, 7.0, 10 mmol/L
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours in the absence of S9-mix, 4 hours in the presence of S9-mix.

NUMBER OF REPLICATIONS:
Duplicate cultures were used for each concentration of the test substance.

GROWTH RATE AND VIABILITY:
On the day of exposure, the growth rate and viability of the cells were checked: the growth rate was 12.3 hours (doubling time), the viability was 96% (expressed as trypan blue exclusion).

DETERMINATION OF CYTOTOXICITY
The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).
Evaluation criteria:
A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 126 mutants per 1,000,000 clonable cells.

A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but smaller than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity and with no evidence of mutagenicity at RTG > 10%, was considered to be an artefact and not indicative of genotoxicity.

The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations.
Statistics:
No statistical analysis was performed.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9-mix the test substance was slightly toxic to the cells
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF CYTOTOXICITY TEST:
In the absence of S9-mix the test substance was slightly toxic to the cells, resulting in a decrease of 24% in relative total growth (RTG). In the presence of S9-mix no toxicity was observed.

CONTROLS:
The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.
Conclusions:
The test substance is considered to be non-mutagenic in this gene mutataion assay.
Executive summary:

In a GLP compliant gene mutation test performed according to OECD guideline 476, potassium tetrafluoroborate was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). One test was conducted. In this test 6 duplicate cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively. The test substance was dissolved in dimethylsuphoxide (DMSO). The highest concentration tested and evaluated for mutagenicity was 10 mmol/L. The test substance was slightly toxic to the cells in the absence of S9 -mix. A slight decrease by 24% of the relative growth (RTG) was observed at the highest dose of 10 mmol/L. In the presence of S9 -mix no cytotoxicity was observed. In both the absence and presence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. All data were within the range of the negative control and the historical background. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; DMSO served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls. It is concluded that under the conditions used in this study, the test substance potassium tetrafluoroborate is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in both the absence and presence of metabolic activation (S9-mix).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Potassium tetrafluoroborate is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions

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:
29 sept 2017 to 28 march 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
As indicated in the EU REACH Regulation (Annex VIII), an in vivo mutagenicity test shall be considered in case of a positive result in any of the in-vitro genotoxicity studies in Annex VII or VIII. For potassium tetrafluoroborate the in-vitro mutagenicity tests show negative results and do not trigger the performance of an in vivo study. It should however be noted that this in vivo mutagenicity study was not performed as part of the EU REACH registration. For this reason no testing proposal for the in-vivo mutagenicity study was submitted.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 8 weeks old at the start of treatment
- Weight at study initiation: 231 ± 8.4 g and the range 214 - 252 g
- Assigned to test groups randomly:yes, under following basis:body weight
- Fasting period before study: Feed was withheld 3 to 4 hours prior to dosing until 1 to 1.5 hours after administration of Potassium tetrafluoroborate.
- Housing: The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm or type MIII height
180 mm, length 380 mm and width 220 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper bedding (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.
- Diet (e.g. ad libitum): The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
- Water (e.g. ad libitum): The animals had free access to tap-water.
- Acclimation period: 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained

IN-LIFE DATES: From: 13 nov 2017 To 15 dec 2017
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. Potassium tetrafluoroborate was suspended in corn oil. The specific gravity of corn oil is 0.9 g/mL. Potassium tetrafluoroborate concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. Potassium tetrafluoroborate concentrations were dosed within 3 hours after preparation.
Duration of treatment / exposure:
Two treatments were performed, administered at a 24-hour interval.
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Positive control(s):
5 animals per dose
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed using O2/CO2. 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 4 mL of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.

DETAILS OF SLIDE PREPARATION:
The supernatant was removed with a Pasteur pipette. Approximately 500 µL 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.

METHOD OF ANALYSIS:
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 cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

OTHER:
To prevent bias, all slides were randomly coded before examination. An adhesive label with 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. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analysed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
Statistics:
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 all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) 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 (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
In case the micronucleus data is not normally distributed the data will be transformed by using the formula y = 1/y. Thereafter the Dunnett’s test will be performed. In case of inhomogeneous variances the the Welch t test with Bonferonni-Holm Adjustment will be applied.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the dose range finding test, three males and three females were dosed with 2000 mg Potassium tetrafluoroborate/kg body weight. The animals were lethargic, had a rough coat and a hunched posture after dosing. Since the test item showed observable toxic effects at a dose level of 2000 mg/kg body weight a full study is necessary according to the guidelines.

RESULTS OF DEFINITIVE STUDY
Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg body weight were selected as appropriate doses for the micronucleus main test. Since there were no differences between sexes in toxicity only male animals were used in the main study.
Five male animals were used in each treatment group. Three additional male animals, treated with 2000 mg Potassium tetrafluoroborate/kg body weight, were used for blood sampling.

Mortality and Toxic Signs
The animals of the groups treated with 500 and 1000 mg Potassium tetrafluoroborate/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
The following clinical observations were made in the groups treated with 2000 mg Potassium tetrafluoroborate/kg body weight:
Within 2.5 hours after the first dosing the animals showed no reaction to treatment.
Within 22 hours after the first dosing four animals had a rough coat and a hunched posture.
Within 2.5 hours after the second dosing two animals still had no reaction to treatment. Six animals were lethargic, had a rough coat and a hunched posture. Within approximately 22 hours after the second dosing five animals had a rough coat and a hunched posture. In addition, three of these animals were lethargic.

Micronucleated Polychromatic Erythrocytes
The mean number of micronucleated polychromatic erythrocytes scored in Potassium tetrafluoroborate treated groups were compared with the corresponding solvent control group.
No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Potassium tetrafluoroborate treated animals compared to the vehicle treated animals. One animal of the lowest dose group showed a high incidence of 12 micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes. Since this high incidence was only observed in one out of five animals of the lowest dose group and the mean number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes of this group was within the 95% control limits of the distribution of the historical negative control database, it was considered not biologically relevant. Moreover, it is not likely that the lowest dose would give a positive response with no positive response in the high dose.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database.
Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

Ratio Polychromatic to Normochromatic Erythrocytes
The groups that were treated with Potassium tetrafluoroborate and the group that was treated with cyclophosphamide 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.
Conclusions:
Potassium tetrafluoroborate is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions
Executive summary:

In a GLP-compliant erythrocyte micronucleus test, according OECD guideline 474, male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg Potassium tetrafluoroborate per kg body weight. In addition three satellite animals were treated with 2000 mg Potassium tetrafluoroborate per kg body weight for blood sampling. A positive control group was dosed once by oral gavage with 20 mg cyclophosphamide (CP) per kg body weight. In total 5 treatment groups were used, each consisting of at least 5 animals. The animals treated with 2000 mg Potassium tetrafluoroborate per kg body weight showed the following toxic signs after dosing: rough coat and a hunched posture. In addition, 6 animals were lethargic. No treatment related clinical signs or mortality were noted in animals treated with 500 and 1000 mg Potassium tetrafluoroborate per kg body weight or control animals receiving vehicle or cyclophosphamide. Bone marrow was sampled 48 hours after the first dosing.

No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Potassium tetrafluoroborate treated animals compared to the vehicle treated animals. One animal of the lowest dose group showed a high incidence of 12 micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes. Since this high incidence was only observed in one out of five animals of the lowest dose group and the mean number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes of this group was within the 95% control limits of the distribution of the historical negative control database, it was considered not biologically relevant. Moreover, it is not likely that the lowest dose would give a positive response with no positive response in the high dose.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met. The groups that were treated with Potassium tetrafluoroborate and the group that was treated with cyclophosphamide 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.

In conclusion, Potassium tetrafluoroborate is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria

Since no in vitro gene mutation study in prokaryotic cells is available for potassium tetrafluoroborate, the results from the structural analogue tetrafluoroboric acid (42%) are used instead (for details see Read-across justification as attached in section 13).

An Ames test with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli strain WP2uvrA was available for tetrafluoroboric acid (42% aqueous solution) (Shimizu, 1985). The study was conducted according to the Guidelines of the Japanese Ministry of Labour. The strains were treated with test concentrations ranging from 1 to 5000 μg/plate, both in the presence and absence of metabolic activation. No increased number of revertants was observed in any case. Based on the results, the substance was concluded to give negative results in the Ames test.

In vitro gene mutation study in mammalian cells

In a GLP compliant gene mutation test performed according to OECD guideline 476, potassium tetrafluoroborate was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix) (TNO Triskelion BV, 2012). One test was conducted. In this test 6 duplicate cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively. The test substance was dissolved in dimethylsuphoxide (DMSO). The highest concentration tested and evaluated for mutagenicity was 10 mmol/L. The test substance was slightly toxic to the cells in the absence of S9 -mix. A slight decrease by 24% of the relative growth (RTG) was observed at the highest dose of 10 mmol/L. In the presence of S9 -mix no cytotoxicity was observed. In both the absence and presence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. All data were within the range of the negative control and the historical background. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; DMSO served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls. It is concluded that under the conditions used in this study, the test substance potassium tetrafluoroborate is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in both the absence and presence of metabolic activation (S9-mix).

In vitro Micronucleus test

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) (TNO Triskelion BV, 2012). 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.

In vivo Micronucleus test

A GLP-compliant erythrocyte micronucleus test was performed according OECD guideline 474. Four groups of 5 male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg Potassium tetrafluoroborate per kg body weight. A positive control group (cyclophosphamide) was included. It was concluded that, under the conditions used in this study, potassium tetrafluoroborate did not induce chromosomal damage or damage to mitotic apparatus of the bone marrow target cells of male mice up to a dose of 2000 mg/kg.


Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity studies, the test substance does not need to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008