Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.