Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-20 to 2012-05-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2008-05-31
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Potassium hexafluoro titanate
- Physical state: solid colourless powder
- Storage condition of test material: in containers tightly closed in a dry, cool and well-ventilated place

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: males: 31 - 32 days; females: 32 - 33 days
- Weight at study initiation: males: 88 - 122 g; females: 83 - 101 g
- Assigned to test groups randomly: yes
- Fasting period before study: feeding was discontinued approx. 16 hours before administration; only tap water was then available ad libitum.
- Housing: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The animals were kept in groups of 2 - 3 by sex in MAKROLON cages (type III plus).
- Diet (ad libitum, except for fasting period before administration): Commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Drinking water
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used:0.8% aqueous hydroxypropylmethylcellulose (Methocel; batch no. 11 A 27-N27, Fagron GmbH & Co., 22885 Barsbüttel, Germany)

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended to the appropriate concentrations in 0.8% aqueous hydroxypropylmethylcellulose . The administration volume was 20 mL/kg b.w.
Duration of treatment / exposure:
single dose application
Frequency of treatment:
once
Post exposure period:
24 hours or 48 hours after the last treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
31, 62 and 125 mg potassium hexafluoro titanate
Basis:
actual ingested
No. of animals per sex per dose:
5 males/5 females per sampling interval and group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (batch no. 068K1131, SIGMA-ALDRICH CHEMIE GmbH, 82024 Taufkirchen, Germany)
- Route of administration: intraperitoneal
- Vehicle: 0.9% NaCl solution (batch no. 12013451; B. Braun Melsungen AG, 34212 Melsungen, Germany)
- Dose level: 27 mg/kg bw
- Administration volume: 20 mL/kg b.w.

Examinations

Tissues and cell types examined:
The slides were coded and randomised before microscopic analysis. Two thousand polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 1000 erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION (RANGE FINDER):
The dose levels had been selected based on a preliminary oral acute toxicity study employing one rat (Crl: CD(SD)) per sex and dose. Five dose levels of 31, 62, 125, 250 and 500 mg potassium hexafluoro titanate/kg b.w. were tested by gavage. The administration volume was 20 mL/kg bw and the vehicle was 0.8% aqueous hydroxypropylmethylcellulose. The animals were observed for symptoms for a period of 3 days.

TREATMENT AND SAMPLING TIMES:
The dose levels should cover a range from the maximum toxicity to little or none. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. The highest dose may also be defined as the dose that produces some indication of toxicity of the bone marrow (e.g. reduction of the proportion of immature erythrocytes among total erythrocytes in the bone marrow) for solutions the maximum feasible application volume.
For the main study, the following sampling times were used: 24 hours for all groups and 48 hours after administration for the vehicle control and the high dose group. The experiment consisted of the following groups, as can been seen in table 1 (please refer to "Any other information on materials and methods incl. tables").

DETAILS OF SLIDE PREPARATION:
The rats were sacrificed at the indicated time points. After having removed some of the muscles, the femurs were excised below the knee and at the iliac joint. The bone marrow was flushed out with calf serum and centrifuged at 850 x g for 3 to 5 minutes. The supernatant was removed and the sediment resuspended in a drop of calf serum by using a Pasteur Pipette. Then a smear of 30 to 60 mm length was prepared.
Once dry, the preparations were immediately fixed in methanol for 5 minutes. Cells were stained for 6 minutes using filtered Mayers Haemaleum. The slides were rinsed with cold tap water for 5 minutes and then further stained in 0.5% w/v ethanolic eosin solution for 1 minute. The slides were again left to air-dry before being dipped in xylene and mounted.

ACCEPTANCE CRITERIA:
The micronucleus test was considered valid if
i) the heterogeneity chi-square test provided evidence of acceptable variability between animals within a group
ii) the incidence of micronucleated PCE in the vehicle control groups fell within or close to the historical vehicle control range
iii) at least 5 analysable animals per sex of each group at each kill were available for analysis
iv) the positive reference chemical (CPA) induced clear and statistically significant increases in the frequencies of micronucleated PCE.
Evaluation criteria:
The test chemical was considered as clearly positive in this assay if:
i) a statistically significant increase in the frequency of micronucleated PCE occurred for at least one dose at one kill time
ii) the frequency of micronucleated PCE at such a point exceeded the historical control range
iii) corroborating evidence was obtained, for example, increased but statistically insignificant frequencies or micronucleated PCE at other doses or kill times, or dose response profiles.
Statistics:
After completion of scoring and decoding of slides, the ratio of PCE/NCE for each animal and the mean for each group was calculated. The individual and group mean frequencies of micronucleated PCE/1000 were also determined.
PCE/NCE ratios were determined in order to evaluate possible bone marrow toxicity.
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971)* as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity test data).
The PCE/NCE ratios and frequencies of micronucleated PCE in the vehicle control animals were compared with historical control ranges to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity chi-square test.
The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in the vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p ≤ 0.05 were accepted as significant.

* References:
- COLQUHOUN, D. Lectures on Biostatistics, Clarendon Press, Oxford (1971)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Please refer to "Additional information on results" below
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Treatment with 31, 62 or 125 mg potassium hexafluoro titanate/kg bw, p.o. revealed slight signs of toxicity. Dose levels of 250 or 500 mg/kg b.w. caused slight signs of toxicity or slight to moderate signs of toxicity and death of all treated animals (death 24 hours after administration or death 1 hour after administration).
Hence, three ascending doses of 31, 62 and 125 mg potassium hexafluoro titanate/kg b.w. were employed.

RESULTS OF DEFINITIVE STUDY
- Clinical signs:
the animals treated with 31 or 62 mg potassium hexafluoro titanate/kg b.w., p.o. revealed pilo-erection 60 or 30 minutes to 3 hours after administration, respectively. Treatment with 125 mg potassium hexafluoro titanate/kg b.w., p.o. caused slightly reduced motility, slight ataxia and pilo-erection 30 minutes to 3 hours and slightly reduced muscle tone and slight dyspnoea 30 to 60 minutes after administration (sacrifice after 24 or 48 hours).
Immediately after sacrifice, bone marrow smears were prepared.
- Micronucleus assay:
No test item-related increase of micronucleated polychromatic erythrocytes was observed in the treated groups as compared to the corresponding vehicle control group at the two sampling times )please refer to table 1 in "Any oterh information on results incl. tables" below). Systemic exposure was demonstrated by signs of little to maximum tolerable toxicity.
The positive reference item group which received cyclophosphamide (27 mg/kg b.w., i.p.) exhibited a significant increase in the number of micronucleated polychromatic erythrocytes.

VALIDITY OF STUDY:
All criteria for validity were met. The data of the positive control were heterogeneous, however, this significance is scientifically not relevant as the data were clearly positive.

STATISTICAL ANALYSIS OF DATA:
The numbers of micronucleated PCE was not influenced by the treatment with the test item. Neither the PCE/NCE ratios nor the numbers of micronucleated PCE were influenced by administration of the test item. No statistical significance was reached after statistical analysis by chi2 test:
The numbers of micronucleated PCE of the negative control and treatment groups were similar to those seen in historical controls.
In all of the cyclophosphamide-treated rats, the numbers of micronucleated PCE significantly exceeded those seen in the vehicle control groups, such that the group mean frequency for both sexes combined (20.5/1000) was approximately 34 times greater than the group mean frequency seen in the concurrent vehicle control.

Any other information on results incl. tables

Table 1 Summarised data of the mutagenicity study

Potassium hexafluoro titanate [mg/kg bw, p.o.]

Sampling time (h)

No. of polychromatic erythrocytes scored per group*

Ratio

PCE/NCE**

Micronucleated polychromatic erythrocytes

 

Mean frequency per 1000 PCE

Significance

Males

Females

Mean*

Males

Females

Mean*

0

24

20000

0.91

0.85

0.88

0.5

0.6

0.6

-

31

24

20000

0.90

0.75

0.83

0.9

0.4

0.7

n.s.

62

24

20000

0.88

0.99

0.94

0.9

0.2

0.6

n.s.

125

24

20000

0.96

0.96

0.96

0.3

0.6

0.5

n.s.

0

48

20000

0.96

0.94

0.95

0.5

0.7

0.6

-

125

48

20000

0.91

0.93

0.92

0.6

0.6

0.6

n.s.

Cyclophosphamide

27 mg/kg bw, i.p.

24

20000

0.53

0.52

0.53

24.6

16.4

20.5

s.

* = males and females combined

** = per 1000 counted cells

s. = significant at p ≤ 0.05 for increases compared to the control

n.s. = not significant at p≤ 0.05 for increases compared to the control

p.o. = per oral

i.p. = intraperitoneal

PCE = ploychromatic erythrocytes

NCE = normochromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Potassium hexafluoro titanate tested up to the maximum tolerated dose of 125 mg potassium hexafluoro titanate/kg bw by oral administration showed no mutagenic properties in the rat bone marrow micronucleus study at the two tested sampling times of 24 hours and 48 hours. Systemic exposure was demonstrated by signs of little to maximum tolerable toxicity.
In the same system, cyclophosphamide induced significant increases in micronucleus frequency, at a decrease of the PCE:NCE ratio of 40%.