Dihydrogen hexafluorozirconate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Read-across from the salt K2ZrF6 to the acid H2ZrF6. bacterial reverse mutation test: negative in vitro mammalian cell gene mutation assay: negative in vitro mammalian cell micronucleus test: positive in vivo micronucleus test in rats: negative Overall conclusion on the genetic toxicity of dihydrogen hexafluorozirconate: negative

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2008-05-31

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Type of assay:

micronucleus assay

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: males: 31 - 32 days; females: 32 - 33 days
- Weight at study initiation: males: 86 - 112 g; females: 80 - 106 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

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

Remarks:

Doses / Concentrations:
62, 125, and 250 mg potassium hexafluoro zirconate/kg bw
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
- Dose level: 27 mg/kg bw
- Vehicle: 0.9% NaCl solution (Batch no. 12013451; B. Braun Melsungen AG, 34212 Melsungen, Germany)
- Administration volume: 20 mL/kg bw

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. Seven dose levels of 8, 16, 31, 62, 125, 250 and 500 mg potassium hexafluoro zirconate/kg bw 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 be 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.

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

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
No signs of toxicity were noted at 8 or 16 mg potassium hexafluoro zirconate/kg bw, p.o. Treatment with 31, 62, 125 or 250 mg potassium hexafluoro zirconate/kg bw, p.o. revealed slight signs of toxicity. The dose level of 500 mg/kg b.w. caused slight to moderate signs of toxicity and death of both animals (1 or 6 hours after administration).
Hence, three ascending doses of 62, 125 and 250 mg potassium hexafluoro zirconate/kg bw were employed.

RESULTS OF DEFINITIVE STUDY
- Clinical signs:
The animals treated with 62 mg potassium hexafluoro zirconate/kg b.w., p.o. revealed pilo-erection 60 minutes to 3 hours after administration. Treatment with 125 or 250 mg potassium hexafluoro zirconate/kg b.w., p.o. caused slightly reduced motility, slight ataxia, slightly reduced muscle tone, slight dyspnoea and pilo-erection 30 minutes to 3 or 6 hours after administration, respectively (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 other 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 negative control, the intermediate dose level and the positive control were heterogeneous, however, this significances are scientifically not relevant due to the small number of micronuclei (negative control and intermediate dose level) and as the data of the positive reference 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 (16.0/1000) was approximately 40 times greater than the group mean frequency seen in the concurrent vehicle control.

Table 1 Summarised data of the mutagenicity study

Potassium hexafluoro zirconate [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	0.68	0.69	0.69	0.4	0.4	0.4	-
62	24	20000	0.77	0.86	0.82	0.5	0.2	0.4	n.s.
125	24	20000	0.95	0.86	0.91	0.4	1.1	0.8	n.s.
250	24	20000	1.05	0.81	0.93	0.7	0.9	0.8	n.s.
0	48	20000	0.89	0.73	0.81	0.2	0.4	0.3	-
250	48	20000	0.88	0.87	0.88	0.5	0.4	0.5	n.s.
Cyclophosphamide
27 mg/kg bw, i.p.	24	20000	0.65	0.47	0.56	20.8	11.1	16.0	s.

* = males and females combined

** = per 1000 counted cells

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

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

p.o. = per oral

i.p. = intraperitoneal

PCE = ploychromatic erythrocytes

NCE = normochromatic erythrocytes

Conclusions:

Interpretation of results (migrated information): negative
Potassium hexafluoro zirconate tested up to the maximum tolerated dose of 250 mg potassium hexafluoro zirconate/kg b.w. 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 (positive reference item) induced significant increases in micronucleus frequency.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Read-across is applied from the salt K2ZrF6 to the acid H2ZrF6. This read-across is justfied based on the chemical similarity of these two substances. The salt K2ZrF6 is soluble in water and dissociates into inorganic ions. H2ZrF6 is an aqueous solution, also containing the respective ions. Under physiological conditions, both substances would yield the same ionic species, which would be responsibel for any toxicological or physological effect. The following paragraphs summarise the results and conclusions for the genetic toxicity of K2ZrF6, and the results are read-across without any restrinctions to the acid H2ZrF6.

The following GLP and guideline conform GLP studies are considered as key studies and will be used for classification:

The in vitro mammalian cell gene mutation test by Lloyd (2012) according to OECD 476 has shown that dipotassium hexafluorozirconate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells up to the highest test concentration, limited by test item precipitation.

Testing in bacteria reverse mutation assays, although of limited relevance for metals (HERAG, 2007), also yielded negative results (Thompson, 1997) up to the limit concentration of 5000 µg/plate.

Negative as well as positive results were obtained in clastogenicity assays: Dipotassium hexafluorozirconate appears to induce micronuclei in vitro in cultured human peripheral blood lymphocytes, but only at high concentrations and via mechanisms that appear to lack physiological relevance (Watters, 2012), and is not mirrored in corresponding in vivo (RL=1) GLP-conform micronucleus test in the bone marrow of rats exposure via the oral route (Flügge, 2012). Availability of the substance was demonstrated by signs of general toxicity, thus exposure of the target organ was ensured.

In conclusion, based upon the evaluation of all available studies, genotoxicity is not an endpoint appropriate to be carried forward to risk characterisation.

 

References:

HERAG (2007) Fact sheet 05 - Mutagenicity. EBRC Consulting GmbH / Hannover /Germany.August 2007. [www.herag.net]

 

Justification for selection of genetic toxicity endpoint
Key study

Justification for classification or non-classification

Based upon the evaluation of all available studies, the classification of dihydrogen hexafluorozirconate with respect to mutagenic potential is not appropriate. Thus, according to Directive EEC 67/548 and to EC Regulation No. 1272/2008, dihydrogen hexafluorozirconate is not considered to have a mutagenic potential, hence, no classification or labelling is required.