Aluminate(1-), tetrafluoro-, cesium (1:1), (T-4)-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 August 1999 (Study Plan) to 29 November 1999 (GLP compliance statement)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

EC Number:

604-086-6

Cas Number:

138577-01-2

Molecular formula:

Al.Cs.F

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Cesium Fluoro Aluminate Complex
- Purity: 99.5%
- Batch: 604/013
- Physical state: White amorphous powder
- Storage condition of test material: At room temperature in the dark
- Expiration date of the lot/batch: 22 May 2001

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: Young adult animals were selected (6 weeks old).
- Number of animals: 5 male and 5 female mice per sampling time in each treatment group.
- Weight at study initiation: 30-34 g (male); 25-27 g (female).
- Assigned to test groups randomly: yes
- Fasting period before study: Feed was withheld 3-4 h prior to dosing until administration of CsAlF-complex.
- Housing: Five animals per sex per group were housed in labelled polycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by B.M.I., Helmond, The Netherlands). In addition, paper bedding was provided as nest material (B.M.I., Helmond, The Netherlands).
- Diet: The animals had free access to standard pelleted laboratory animal diet (Carfil Quality BV, Oud-Turnhout, Belgium)
- Water: The animals had free access to tap-water.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% (w/v) carobxymethylcellulose (Genfarma B.V., Maarsen, The Netherlands)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test subsatnce was suspended in 1% (w/v) carboxymethylcellulose. Test substance concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. Test substance concentrations were dosed within 4 hours after preparation.
The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of CsAlF-complex. The dosing volume was 10 ml/kg body weight.

Duration of treatment / exposure:

Dose range finding study: The following dose groups were administered as single dose to select the adequate dose range for the Micronucleus test:
- 2000 mg/kg bw: 2 males and 2 females;
- 1000 mg/kg bw: 3 males and 3 females;
- 750 mg/kg bw: 3 males.
The study duration was three days. During this period mortality and physical condition were recorded daily.

Main study: Five male and five female mice were used per sampling time in each treatment group. The animals were dosed once and sampled according to the following scheme:
- CsAlF-complex: 1000 mg/kg bw; sampling time 24 and 48 hours;
- CsAlF-complex: 500 mg/kg bw, sampling time 24 and 48 hours;
- CsAlF-complex: 250 mg/kg bw, samping time 24 and 48 hours;
- Cyclophosphamide: 50 mg/kg bw, sampling time 48 hours.

Frequency of treatment:

Single dose.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Dose range finding study: 750, 1000 and 2000 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Main study: 250, 500 and 1000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

Range finding study :
- 2000 mg/kg bw: 2 male and 2 female
- 1000 mg/kg bw: 3 males and 3 females
- 750 mg/kg bw: 3 males

Main study:
- 5 male and 5 female mice per sampling time in each treatment group.

Control animals:

no

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline dosed as a single oral intubation of 50 mg/kg body weight.

Examinations

Tissues and cell types examined:

At necropsy, bone marrow cells of both femurs were collected from each mouse. The bone marrow cells were immediately collected into foetal calf serum and processed into glass drawn smears according to the method described by Schmid (1977). Two bone marrow smears per animal were prepared.

Details of tissue and slide preparation:

ISOLATION OF BONE MARROW:
Bone marrow of the groups treated with CsAlF-complex was sampled 24 or 48 hours after dosing. Bone marrow of the positive control group was isolated 48 hours after dosing. The animals were sacrificed by cervical dislocation. 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 2 ml of fetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm for 5 min.

PREPARATION OF BONE MARROW SMEARS
The supernatant was removed with a Pasteur pipette. A drop of 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/ether and cleaned with a tissue and marked (study number and 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 and air-dried overnight. Two slides were prepared per animal.

STAINING OF THE BONE MARROW SMEARS
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer. The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

ANALYSIS OF THE BONE MARROW SMEARS FOR MICRONUCLEI
To prevent bias, all slides were randomly coded before examination. 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 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, two-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes in the combined data for both sexes or in the data for male or female groups separately.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: At 2000 mg/kg bw, one female and one male animal died within 2 days after dosing. No other mortality occurred. At 1000 mg/kg, hunched posture and rough coat were observed within 30 minutes after dosing and these effects persisted in 2 animals on Day 2 and 3. At 500 mg/kg bw, hunched posture and rough coat was only observed in one animal.

RESULTS OF DEFINITIVE STUDY
- Doses: 250, 500 and 1000 mg/kg bw
- Mortality and toxic signs:
The animals of the groups treated with 250 and 500 mg/kg bw and the animals of the positive control group showed no abnormalities. The following clinical observations were made in the groups treated with 1000 mg CsAlF-complex/kg bw: during the first hour after dosing all animals had a rough coat. Nine male animals also had a hunched posture and five of them were lethargic. One female animal also had a hunched posture and was lethargic. Within 18 hours after dosing two animals still had a rough coat and a hunched posture. All other animals recovered from the treatment. Within 42 hours after dosing the animals showed no reaction to treatment.
- Micronucleated polychromatic erythrocytes:
The mean number of micronucleated polychromatic erythrocytes scored in CsAlF-complex treated groups were compared with the corresponding historical solvent control data. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test substance treated animals. The incidence of micronucleated polychromatic erythrocytes of all treated animals were between or equal to the minimum and maximum value of the historical control data range.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, the acceptability criteria of the test were met.
- Ratio polychromatic to normochromatic erythrocytes:
The animals of the groups, which were treated with CsAlF-complex and the positive control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of the test substance on the erythropoiesis.

Applicant's summary and conclusion

Conclusions:

Cesium Fluoro Aluminate Complex is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 1000 mg/kg bw under the experimental conditions described in this report.

Executive summary:

Cesium fluoro aluminate complex was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. The test was performed according to the OECD Testing Guideline 474 and under GLP.

Six groups comprising 5 males and 5 females, received a single oral intubation. Two groups (A and B) were dosed with 1000 mg/kg body weight, two groups (C and D) were dosed with 500 mg/kg body weight, two groups (E and F) dosed with 250 mg/kg body weight. One group (G) treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow was sampled 24 or 28 hours after dosing. Bone marrow from the positive control group (G), was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with cesium fluoro aluminate complex.

The groups that were treated with cesium fluoro aluminate complex and the positive control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes (in controls in the region of 1), which reflects a lack of toxic effects of this compound on the erythropoiesis.

It is concluded that cesium fluoro aluminate complex is not mutagenic in the micronucleus test under the experimental conditions described in this report.