Tin difluoride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The reference shows several shortcomings and is not considered compliant with the study design as foreseen in the OECD 474: (i) Adult animals were used instead of young animals as foreseen in the OECD guideline (ii) Single dose treatment is appropriate in case a limit does of 2000mg/kg bw is administered, however the animals were exposed to a single dose of max. 15µg/kg bw which does not allow an analysis of a dose-response relationship. (iii) cytotoxicity not determined; hence correlation of DNA damage and cytotoxicity cannot be performed. (iv) the number of animals per dose group is too low in order to demonstrate statistical robustness (v) the experimental procedure is not described in sufficient detail in order to e.g. determined the number of cells examined per animal (vi) the evaluation of the comets by comparing tail length with nucleus diameter is unusual, commonly tail length, %DNE in tail or tail moment is determined. Based on the above given shortcomings and the missing GLP status, the reference is considered not reliable and unsuitable for chemicals hazard assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The reference shows several shortcomings and is not considered compliant with the study design as foreseen in the OECD 474: (i) Adult animals were used instead of young animals as foreseen in the OECD guideline. (ii) Single dose treatment is appropriate in case a limit does of 2000mg/kg bw is administered, however the animals were exposed to a single dose of max. 15µg/kg bw which does not allow an analysis of a dose-response relationship. (iii) Target organ exposure by determination of the PCE/NCE ratio or presence of general signs of toxicity was not determined; hence target organ exposure could not be demonstrated. (iv) The number of animals per dose group is too low in order to demonstrate statistical robustness; the guideline foresees at least 5 animals per dose and sex. Based on the above given shortcomings and the missing GLP status, the reference is considered not reliable and unsuitable for chemicals hazard assessment.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The clastogenic effect of stannous chloride dihydrate in rats was observed. Male rats (5 animals per dose group) were given a 100µL single intravenous injection of 500 µg/mL (equivalent to 50 µg per animal and 12.5-15 µg/kg bw). Negative control animals received 0.9% NaCl solution, positive control animals received 50 mg/kg cyclophosphamide via i.v. injection. Animals were sacrificed 36 hours post exposure, femora dissected, the bone marrow extracted and the cells were stained. A total of 2000 polychromatic erythrocytes were analysed per animal.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fundaçao Instituto Oswaldo Cruz (Rio de Janeiro, Brazil)
- Age: 3 months
- Weight: 250 - 300 g
- Assigned to test groups randomly: yes
- Housing: housed in polypropylene cages (six rats per cage) with hardwood-chip bedding
- Diet (ad libitum)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 24 ± 1°C
- Humidity: 55 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intravenous

Vehicle:

- Vehicle(s)/solvent(s) used: NaCl 0.9%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was dissolved in NaCl 0.9% solution.
Animals were administered a single 100 µL intravenous injection of the test item in the vehicle.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

once

Dose / conc.:

500 other: µg/mL injected intravenously

No. of animals per sex per dose:

5 rats

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: single 100 µL intravenous injection
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Bone marrow cells were analyzed by 1000× magnification (immersion objective). Two-thousand polychromatic erythrocytes (PCE’s) from each animal (1,000 on each slide) were analyzed.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES
The animals were sacrificed thirty-six hours following the start of treatment with the tested agents; the femur was excised and bone marrow cells were obtained. The bone marrow cells were placed in tubes. The cell suspension was centrifuged for 5 minutes, at 1,000 rpm, and the supernatant was discarded. Cell viability evaluation was performed with trypan blue following centrifuging, so that all samples would have the same cell concentration at the moment of smear preparation (1 × 10^6 cells/mL).

DETAILS OF SLIDE PREPARATION:
Smear preparation and slide dyeing a drop of cell suspension was placed onto a slide, smearing being performed by means of another slide, two slides being made per animal. Slides were left at room temperature to dry, being later fixed in methanol for 10 minutes, and left at room temperature for drying.
Slide dyeing was carried out with the Giemsa dye diluted in PBS (1:10), for 15 min. Following dyeing, slides were washed with distilled water for excess dye removal, being left to dry once more at room temperature.

Evaluation criteria:

no data

Statistics:

To evaluate the toxic effects of testing agents, data were analyzed using the ANOVA and Tukey–Kramer multiple comparison tests by statistical program InStat version 3.01 (GraphPad Software, San Diego, CA, USA). Significance level (p < 0.05) was adopted to compare data within the experiment.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

- Positive control: micronucleated bone marrow polychromatic erythrocytes (MNPCE) means (per 1,000 PCE) in rats were 16.2 (p < 0.05).
- SnCl2 (500 μg/mL): no formation of micronucleus in bone marrow. The MNPCE means were 2.2 (p < 0.05). Treatment did not exhibit a significant difference when compared to the negative control group (p > 0.05).

Conclusions:

Stannous chloride did not show an increase in the MN-PCE frequency. Based on the results, stannous chloride is considered non-clastogenic in the bone marrow of i.v. exposed rats.

Data source

Reference

Reference Type:

publication

Title:

Evaluation of deoxyribonucleic acid toxicity induced by the radiopharmaceutical 99mTechnetium-Methylenediphosphonic acid and by stannous chloride in Wistar rats

Author:

De Mattos, J.C.P. et al.

Year:

2012

Bibliographic source:

Molecules 17: 12974-12983.

Materials and methods

Principles of method if other than guideline:

The DNA damaging effect via Comet assay of stannous chloride dihydrate in rats was observed. Male rats (5 animals per dose group) were given a 100µL single intravenous injection of 500µg/mL (equivalent to 50 µg per animal and 12.5-15 µg/kg bw). Negative control animals received 0.9% NaCl solution, positive control animals received 50mg/kg cyclophosphamide via i.v. injection. Blood was collected before the treatment and 3 and 24 hours thereafter. Cells were treated directly, transferred to LMP agarose gels, incubated in a lysis solution and subsequently in alkaline electrophoresis buffer. Comets were evaluated via a four class rating (no migration to tail two-time longer than nucleus), 50 nuclei were evaluated.

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

tin chloride dihydrate

IUPAC Name:

tin chloride dihydrate

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Stannous chloride (obtained from Sigma (St Louis, MO, USA))
- Molecular formula (if other than submission substance): SnCl2 * 2H20

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fundaçao Instituto Oswaldo Cruz (Rio de Janeiro, Brazil)
- Age: 3 months
- Weight: 250 - 300 g
- Assigned to test groups randomly: yes
- Housing: housed in polypropylene cages (six rats per cage)with hardwood-chip bedding
- Diet (ad libitum)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 24 ± 1°C
- Humidity: 55 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intravenous

Vehicle:

- Vehicle(s)/solvent(s) used: NaCl 0.9%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was dissolved in NaCl 0.9% solution.
Animals were administered a single 100 µL intravenous injection of the test item in the vehicle.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

once

Doses / concentrations

Dose / conc.:

500 other: µg/mL (injected intravenously)

No. of animals per sex per dose:

6 rats

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Examinations

Tissues and cell types examined:

Slides were stained and immediately evaluated at 400x magnification, using a fluorescence microscope equipped with a 580 nm excitation filter and a 590 nm barrier filter set, and quantified.
Damages were assigned based on the visual aspect of the comets, considering the DNA migration pattern, and cells were scored, according to tail length, into four classes (0–3). Class 0 means undamaged nucleus, there being no tail formation. In class 1, the nucleus displays a short tail, smaller than nucleus diameter; class 2, tail length is between 1 and 2 times head diameter. Nucleus maximally damaged is allocated to class 3, where tail is two times, or longer, than nucleus diameter. For each slide, 50 nuclei were randomly observed, always from the left to the right side, total scoring (TS) being obtained by multiplying the number of cells in each class (nx) by the damage class (Equation 1), being ranged from 0 to 150:

TS = (0 x n0) +(1 · n1) + (2 · n2) + (3 · n3) (Equation 1)

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES
The animals were sacrificed in a CO2 chamber thirty-six hours after the treatment.

Blood was collected from all rats immediately before intravenous injections and at 3 and 24 hours after chemical solution administration.

METHOD OF ANALYSIS/DETAILS OF SLIDE PREPARATION:
Following the treatments, 10 μL total blood aliquots from each animal were mixed with 120 μL prewarmed (37 °C) 0.5% low melting point (LMP) agarose, in PBS, and the whole volume put on a microscope slide coated with 1.5% normal agarose, in PBS. Ten slides from each animal, for each time interval studied, were prepared as described above, covered with microscope cover slips and kept at 4°C for 20 minutes, to accelerate gelling. Following agarose, slides were submerged for 1 hour in a freshly-prepared cold lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 10% DMSO, 1% Triton X-100, pH 10.0). Following that, slides were transferred to an electrophoresis chamber containing a high pH (>13) buffer (300 mM NaOH, 1 mM EDTA) and incubated for 20 minutes, at room temperature, to allow the DNA to unwind. Electrophoresis was performed at 1.0 V/cm, 300 mA for 25 minutes. Subsequently, slides were washed three times with neutralization buffer (0.4 M Tris-HCl, pH 7.5), fixed in 100% ethanol, washed with ultrapure water (Milli-Q system) and dried at room temperature, overnight. Slides were stained with ethidium bromide (30 μL, 40 μg/mL) and immediately evaluated at 400x magnification, using a fluorescence microscope equipped with a 580 nm excitation filter and a 590 nm barrier filter set, and quantified.

Three independent experiments were carried out for each time period throughout the study, all slides being analyzed.

Evaluation criteria:

no data

Statistics:

To evaluate the toxic effects of testing agents, data were analyzed using the ANOVA and Tukey–Kramer multiple comparison tests by statistical program InStat version 3.01 (GraphPad Software, San Diego, CA, USA). Significance level (p < 0.05) was adopted to compare data within the experiment, where 6 animals per group were used.

Results and discussion

Test results

Sex:

male

Genotoxicity:

other: no conclusive statement can be made

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

- Negative control group: NaCl 0.9% sterile solution was not able to induce a statistically significant increase (p > 0.05) in the number of DNA lesions of rat blood cells.
- Despite the high sensitivity of the technique used, which detects low damage levels, the results show that the condition, under which the rats were submitted during the experiment did not interfere with the parameters analyzed.
- SnCl2 (500 μg/mL): statistically significant increment (p < 0.001) in comet numbers in peripheral blood nuclear cells, 3 hours after exposure was observed. 24 hours after the administration, the number of lesions had already fallen back to values close to those of control.

Applicant's summary and conclusion

Conclusions:

Due to the significant reporting and experimental deficiencies no conclusive statement on the outcome of the study can be made.