Tin dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published data fulfilled basically scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The experimental procedures for the SOS chromtest were mainly based on what was established by Quillard and Hofnung (1985).
The SOS Chromotest is able to detect DNA damage by the measurement of induction of sfiA, which is a gene controlled by the general repressor of the SOS system in E.Coli.

GLP compliance:

no

Type of assay:

other: SOS Chromtest

Species / strain / cell type:

E. coli, other: PQ37

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

The experiment was performed at a concentration of chemical at which a decrease of the activity of alkaline phosphatase was observed.
The test item was used at a concentration ranging from 10 tp 1010^4 µg/plate.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Species / strain:

S. typhimurium TA 1535

Remarks:

Not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1537

Remarks:

Not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 98

Remarks:

Not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 100

Remarks:

Not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

E. coli WP2

Remarks:

Not determinated, PQ37 has been tested

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

E. coli, other:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The enzyme activities of alkaline phosphatase and ß-galactosidase were calculated with the following formula: Enzyme unit = 1000 x A420/t.

A420 is the optical density at 420 nm and it is the reaction time (90 min). Then the ratio ß-galactosidase units to alkaline phosphatase units (R(C)) was calculated and the induction factor (I(C)) was finally calculated thus: I(C) = R(C)/R(0). R(C) represents the value at concentration C, and R(0) is that at concentration 0. This induction factor (I(C)) expresses the SOS-inducing potency of the tested chemical.

When a significant increase in ß-galactosidase units of the tested chemical compared to that of the control was recognized by t-test (p < 0.05) and an enhancement of I(C) according to the increase of treated amount was shown, the chemical is considered as an SOS inducer.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published data fulfilled basically scientific principles.

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION
See attached document

Qualifier:

no guideline followed

Principles of method if other than guideline:

The method named rec-assay is based on the observation of differential growth sensitivities to drugs in wild and recombination-deficient strains of Bacillus subtilis. When a chemical is more inhibitory for Rec- than for Rec+ cells, it is reasonable to suspect mutagenicity based on its DNA-damaging capacity.

GLP compliance:

no

Type of assay:

Bacillus subtilis recombination assay

Species / strain / cell type:

bacteria, other: bacillus subtilis strains H17

Additional strain / cell type characteristics:

other: Rec+, arg- and trp-

Species / strain / cell type:

bacteria, other: bacillus subtilis M45

Additional strain / cell type characteristics:

other: Rec-, arg- and trp-

Metabolic activation:

without

Test concentrations with justification for top dose:

an aliquot of 0.05 ml of the metal solution ( SnCl4 in water, 0.05M) was dropped on to a filter paper disc (diameter 10mm) which had been placed at the starting point of the streaks of Rec+ and Rec- bacteria.

Species / strain:

S. typhimurium TA 1535

Remarks:

not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1537

Remarks:

not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 98

Remarks:

not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 100

Remarks:

not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

E. coli WP2

Remarks:

not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

bacteria, other: bacillus subtilis strains H17

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

bacteria, other: bacillus subtilis M45

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

negative without metabolic activation

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

It's a method building and verification report, the substance stannic chloride and stannous chloride was two of plentiful test substances which were used to verify the effectiveness of the method. Due to no details for the test material (purity, source, concentration, etc.), the reliability of this report is not assignable. However, the results can also give a evidence of the non-mutagenicity for stannic and stannous ions.

Qualifier:

no guideline followed

Principles of method if other than guideline:

the rec-assay is a simple and efficient procedure for screening environmental DNA-damaging chemicals.

GLP compliance:

no

Type of assay:

Bacillus subtilis recombination assay

Species / strain / cell type:

other: bacillus subtilis M45 Rec-

Additional strain / cell type characteristics:

other: Rec45 arg try

Species / strain / cell type:

other: bacillus subtilis H17 Rec+

Additional strain / cell type characteristics:

other: Rec+ arg try

Metabolic activation:

with and without

Metabolic activation system:

Araclor S9

Test concentrations with justification for top dose:

Strains H17 Rec+ and M45 Rec- are grown overnight in B-2 broth (meat wet extract, 10 g; polypeptone dry powder, 10 g; NaC1, 5 g; water, 1000 ml
pH adjusted to 7.0). Each culture is streaked radially on tec “dry” surface of broth agar (15 g/litre of agar added to the liquid broth), and a paper disk (diameter 16 mm) containing the test item test solution (10-10^4 µg/plate) is placed over the starting points of the streaks. The plates are incubated at 37°C for about 20 hr. after which the lengths of the inhibition zones are measured.
The sensitivity of the assay is often increased several fold by first keeping the plates overnight at 4°C to permit diffusion of the test item through the agar and then incubating them overnight at 37°C. The sensitivity of the assay is also increased several fold by using thawed cells that have been previously kept at -80°C in broth culture with 12.5% glycerol supplementation in place of fresh-grown cells.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Species / strain:

S. typhimurium TA 1535

Remarks:

Not determinate, the study has been performed on different strains

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1537

Remarks:

Not determinate, the study has been performed on different strains

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 98

Remarks:

Not determinate, the study has been performed on different strains

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 100

Remarks:

Not determinate, the study has been performed on different strains

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

E. coli WP2

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

other: Various bacteria

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Among 144 metal compounds rec-assayed with cold incubation, SnCl4 resulted to be negative.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Published data fulfilled basically scientific principles, however only 2 strains were used for the test: TA 98 and TA 100.

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION
Inorganic tin (supplied as SnCI4-5H20) has been tested. See document attached.

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study, it has been used a modification of the conventional Salmonella mutagenicity assay (Maron and Ames, 1983), that is, washing the tested strain with phosphate buffer and removing each tested chemical. By this modified method, the mutagenicity of organotin compounds could be estimated.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

The inorganic tin (SnCl4 x 5H2O) was tested in the range from 0.1 to 100 µg/tube.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Species / strain:

S. typhimurium TA 1535

Remarks:

Not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1537

Remarks:

Not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

E. coli WP2

Remarks:

Not determinated

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The colonies of survivors were detected with the method by Nohmi et al. (1986), and the IMF value was determined. The IMF value was calculated by the ratio of His+ revertants to survivors colonies: (IMF value = (Rt — Rc)/St (Rt = His+ revertants on tested chemical, Rc = His+ revertants on the control, St = surviving colonies on tested chemical) (Nohmi et al., 1986).

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

SnCl4 does not show a significant increase of His+ revertants in the assays with both the type of S typhimurium strain TA 100 and TA 98

Reference 5

Endpoint:

genetic toxicity in vitro, other

Remarks:

Type of genotoxicity: other: chromosomal abnormalities, sister-chromatid exchanges (SCEs) and cell cycle kinetics.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published data fulfilled basically scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study inorganic tin chloride were investigated with respect to the induction of chromosomal aberrations, sister-chromatid exchanges and their effects on cell cycle kinetics in human peripheral blood lymphocytes in vitro.

GLP compliance:

no

Type of assay:

other: human white blood cells

Target gene:

not applicable

Species / strain / cell type:

other: human white blood cells

Details on mammalian cell type (if applicable):

5 ml venous blood was collected from 27 healthy normal male voluntary donors 30-40 years of age. These individuals did not smoke and had not been exposed to radiation or drugs. 0.3 ml of heparinized whole blood was inoculated into the culture medium (RPMI-1640, Gibco) containing 20% heat-inactivated human AB serum, phyto-hemagglutinin (Gibco, 0.2 ml/5 ml culture) and 5-bromodeoxyuridine (Sigma, 8 µg/ml).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

The final concentrations used were 20 and 10 µg, and 1 and 0.5µg per 5 ml of culture for stannic chloride and trimethyltin chloride respectively.

Vehicle / solvent:

Culture

Untreated negative controls:

yes

Remarks:

Culture

Negative solvent / vehicle controls:

yes

Remarks:

Culture

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

The chemical to be tested was added just after inoculation. Control and treated cultures were maintained in replicate sets. Two different concentrations of the same chemical were tested on the same sample. Treatment period was 72 h at 37°C in the dark.
After 70 h of inoculation, cells were pretreated with colchicine for 2 h followed by hypotonic treatment in 0.09% NaCl in deionized water preheated at 37°C and fixation in 3:1 methanol:acetic acid. Cells were collected on clean slides, air-dried and stained in Hoechst 33258 (5 µg/ml) followed by photo-illumination in sunlight, mounted in 2 × SSC buffer, washed thoroughly and stained in 2% Giemsa in phosphate buffer for 8-10 min.

Evaluation criteria:

Slides were coded for data entry and observed according to standard procedure. For each ex-perimental set 50, 100 and 150 metaphases were observed for sister-chromatid exchanges (M2), chromosomal abnormalities (M3) and cell cycle kinetics respectively. For cell cycle kinetics, the replicative index (RI) was calculated as RI = 1 M1% + 2 M2% + 3 M3%/100 by scoring first (M1, second- (M2) and third- (M 3) division metaphases from differentially stained plates.

Statistics:

Data were analyzed statistically following Student's t test.

Species / strain:

other:

Remarks:

human white blood cells

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

In comparison to control sets, the two concentrations of both compounds significantly elevated the numbers of chromosomal aberrations and sister-chromatid exchanges in relation to the doses given. The rate of cell generation cycle was lowered to levels directly proportional to the concentrations used. Stannic chloride significantly increased the frequency of chromosomal abnormalities (P < 0.05-P < 0.001) with the higher dose, as compared to that of trimethyltin chloride; however, with the lower dose the effect was similar. Trimethyltin chloride induced a slightly higher frequency of sister-chromatid exchanges. The replicative index was more depressed by stannic chloride with both doses. The first-cycle metaphases (M1) remained at a much higher frequency even after 72 h (two cell cycles).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation High concentration of 20 µg/5 ml culture

Extremely high dose of stannic chloride can induce a much high frequency of chromosomal abnormalities and a great reduction of cell cycle kinetics. The significantly higher clastogenicity of stannic chloride may be related to the much large amounts of the salt used and to the high frequency of inter-individual variation. Inter- and intra-individual variations are important factors in assessing chromosomal damage (Obe and Beek, 1984) and variations in chromosomal aberrations and sister-chromatid exchanges in control populations are well known (Dewdney et al., 1986; Ghosh et al., 1988b; Obeet al., 1984). Therefore the highly increased incidences of chromosome aberrations and SCEs may be due to the much large amounts of the salt used but not to the Tin IV per se.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published data fulfilled basically scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The procedures used for alkaline sucrose gradient analysis and the determination of cell survival have been described in detail (Douglas and Grant, 1980). Briefly, cells (8 x 10^5) were grown in 60-mm tissue culture dishes and labelled for 24 h with [14 C]thymidine (14 C-TdR, Amersham 53 mCi/mM) or [3 H]thymidine (3 H-TdR, Amersham 46 Ci/mM) at 1 µCi/ml. At the end of this labelling period, cells were incubated for 4 h in nonradioactive medium. The cells labelled with 14C-TdR were designated as controls and were treated with solvent (1% M/100 HCl in serum-free MEM). The cells that were labelled with 3H-TdR were similarly treated with 1% solutions of tin(IV) in serum-free MEM for 1 h at 37°C. After the treatment period, the cells were washed 3 times in ice-cold PBS, removed from the dishes with a rubber policeman and resuspended in 1 ml of ice-cold PBS.
Alkaline sucrose sedimentation was carried out according to the method of Palcic (Palcic and Skarsgard, 1972).

GLP compliance:

no

Type of assay:

other: alkaline sucrose gradient analysis

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

the test item was dissolved in 2-3 mil of HCl and passed through a 0.45 µm polycarbonate filter. These solutions were then diluted with deionized distilled water at 0°C to give a stock that was 100 X the final concentration required in the cell suspensions.
Chinese hamster cells were treated with various concentration of the test item ranging from 10 to 550 µM.

Untreated negative controls:

yes

Remarks:

solvent-treated cells were provided for negative control

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: potassium dichromate

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published literature fulfilled basically scientific principles.

Justification for type of information:

CATEGORY APPROACH JUSTIFICATION
Inorganic tin (supplied as Tin chloride / tin(4+) tetrachloride) has been tested. See document attached

Qualifier:

no guideline followed

Principles of method if other than guideline:

To provide information about possible genotoxic effects in man, the DNA damaging effects of in vitro exposure of human white blood cells to tin compounds was studied. Meanwhile the uptake capability of Tin II and Tin IV were also evaluated by human white blood cells (WBS). In addition, the DNA strand break damage of WBS after exposure to Tin II and Tin IV was also examined by fluorometric analysis of DNA unwinding (FADU). In the report it also evaluate the further effects of Tin II and Tin IV on Con A-stimulated DNA synthesis in peripheral blood lymphocytes HPBL and mouse splenocytes.

GLP compliance:

no

Remarks:

Old publication

Type of assay:

other: fluorometric analysis of DNA unwinding (FADU); Con A-stimulated DNA synthesis in peripheral blood lymphocytes HPBL and mouse splenocytes assay

Target gene:

Not applicable

Species / strain / cell type:

other: human white blood cells

Details on mammalian cell type (if applicable):

Human WBC were isolated by diluting 1 vol. of freshly collected blood (3 mM EDTA anticoagulant) with 3 vol. of 0.87% ammonium chloride, 10 mM Tris-HCl (pH 7.2-7.3) and allowing red blood cells to lyse at 0°C. After 20-25 min. WBC were collected by centrifugation (260 g, 0 °C, 15 min) washed once in lysing solution and once in a balanced salt solution (BSS; MgS04•7H2O, 100 mg; KCl, 400 mg; NaCl, 8.0 g; HEPES, 5.96 g; glucose 1.0 g; water to 11; final pH 7.3).

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

HPBL were isolated from heparinized blood samples by Ficoll-Paque (Pharmacia) centrifugation (400 g, 18 °C, 30 min).

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mammalian cell line, other: mouse splenocytes

Details on mammalian cell type (if applicable):

Mouse splenocytes were isolated from C57B6/10 male mice. Spleens were removed and gently pressed through a stainless steel screen (100 mesh) into BSS or RPMI-1640 tissue culture medium (Gibco) at 0 °C. Large clumps of cells and tissue debris were removed by settling for 5 min at 0°C; cells were collected from the supernatant by centrifugation (400 g, 0 °C, 15 min) and resuspended at 2E6/ml in RPMI-1640 without serum.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

Uptake test: 100 μM tin(II) and tin(IV)
FADU assay: Tin II: 5, 10, 25, 50 μM Tin IV: not specified
Con A-stimulated DNA synthesis in peripheral blood lymphocytes HPBL and mouse splenocytes:
HPBL: Tin II, concentration not specified.
mouse splenocytes: Tin II and Tin IV: (5-50 μM)

Vehicle / solvent:

Water

Untreated negative controls:

yes

Remarks:

Cr (III)

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Cr (VI)

Remarks:

FADU test

Details on test system and experimental conditions:

Measurement of tin(II)-113 uptake by human WBC
Tin(II)-113 dichloride (New England Nuclear, 18.2 mCi/mg) was diluted with various amounts of non-radioactive tin(II) dichloride to give 100 concentrated stock solutions containing 10 - 30 μCi/ml. The uptake of tin (II)- 113 into WBC was determined by the method of Strauss et al. Briefly. cells were centrifuged through an oil gradient consisting of Dow Corning silicone fluids, 550:556 (12:13) using a Beckman D-9 microfuge (l2 000 g. 15 s). The tip of each centrifuge tube containing the labelled cells was cut off and assayed for radioactivity in a well-type gamma counter.

Microbeam analysis of human WBC treated with tin(II) and tin (IV)
Human WBC (2E6/ml) in BSS were treated with 100 μM tin(II) or tin(IV) for 15 min at 37°C. Cells .were collected, washed, fixed for 1.5 h in 2.5 % glutaraldehyde, dehydrated through an alcohol series, transferred to propylene oxide and embedded in epon. Embedded cells were stained sequentially (20 min each treatment) in uranyl acetate and lead citrate and then mounted on gold grids. Using a Siemens electron microscope., specific areas of cells were irradiated with a 3 μ Amp beam (0.2-0.5 μm diameter). Radioactivity from the characteristic K-alpha (Kα1 Kα2) and K-beta (KU) tin X-rays were collected for 300s in a multichannel analyzer. This provided a semi-quantitative estimate of the tin concentration in specific areas of the cells.

Fluorometric analysis of DNA unwinding (FADU) for the detection of DNA strand break damage

Human WBC in BSS at the specified cell concentration were treated with various concentrations of tinl(II) and tin(IV) at 0°C or 37% for 30 min. Some cells were exposed to 1 Gy of Co-60 gamma radiation (0.156 Gy/min) at 0 °C to introduce a known number of strand breaks. Other cells were exposed at 37 °C to 50 μM Cr(VI), a known DNA damaging agent and to Cr(III), a negative control, which does not penetrate cells. After treatment, cells were collected by centrifugation (260 g, °C 15 min), washed twice in BSS at 0°C and then assayed for DNA damage by FADU.
The FADU procedure has been described in detail elsewhere. Briefly, cells that have been previously exposed to various agents are gently lysed to liberate very large DNA molecules. An alkaline solution is added and DNA (normally double-stranded in the cell) slowly unwinds to form single-stranded DNA. The unwinding rate is estimated by determining the residual fraction of DNA which has remained in the double-stranded form. For this purpose, a fluorescent dye, ethidium bromide is added, which binds to duplex DNA but not to single stranded DNA or to RNA under the conditions used. From the fluorometric measurements, the percentage of cellular DNA remaining double-stranded after a period of exposure to alkali can be calculated both for control cells (DC) and for treated cells (Dx). The expression, Qd = - 100 log (Dx/Dc) is a measure of the amount of DNA strand breakage induced by a given treatment; for Co-60 gamma radiation, it yields a nearly linear dose-response curve to at least 4 Gy. One Gy is taken to produce 1200 single strand breaks per cell 120,211. The fluorescence change for the tin(II) treatments is converted to single strand breaks/genome by reference to a gamma ray dose response curve.

The effect of tin(II) and tin(IV) on Con A-stimulated DNA synthesis in HPBL and mouse splenocytes
HPBL (1E6/ml in BSS without serum) were treated with various concentrations of tin(II) for 30 min at 37°C. Cells were resuspended in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum (Flow Labs) and 50 μM 2-mercaptoethanol and then stimulated to undergo DNA synthesis by the addition of 20 Kg/ml Con A (Sigma Chemical Company). The cells were distributed in quadruplicate to 12 × 75 mm Falcon culture tubes and then incubated at 37°C in an atmosphere of 5% CO2, 95% air and high relative humidity. After 96 hours, cells were labelled for 1 h with 1 μCi/ml [methyl-3H]thymidine (Amersham, 50 Ci/mmol) and viability was determined microscopically by trypan blue exclusion. Incorporation of 3H into an acid insoluble fraction was determined by collecting cells onto GF/C filters and washing sequentially with 30 ml phosphate buffered saline (pH 7.4), 5%, trichloroacetic acid and 95% ethanol, all at 0°C.
Mouse splenocytes were treated with various concentrations of tin(I1) and tin(IV) (5-50 μM) and then stimulated with Con A (2 Kg/ml) and cultured in the same way, except that the rate of DNA synthesis was monitored after 72 h of incubation.

Evaluation criteria:

Measurement of tin(II)-113 uptake by human WBC
The tip of each centrifuge tube containing the labelled cells was cut off and assayed for radioactivity in a well-type gamma counter.

Microbeam analysis of human WBC treated with tin(II) and tin (IV)
Using a Siemens electron microscope., specific areas of cells were irradiated with a 3 μ Amp beam (0.2-0.5 μm diameter). Radioactivity from the characteristic K-alpha (Kα1 Kα2) and K-beta (KU) tin X-rays were collected for 300s in a multichannel analyzer. This provided a semi-quantitative estimate of the tin concentration in specific areas of the cells.

Fluorometric analysis of DNA unwinding (FADU) for the detection of DNA strand break damage
The expression, Qd = - 100 log (Dx/Dc) is a measure of the amount of DNA strand breakage induced by a given treatment.

Statistics:

no data

Species / strain:

other: human white blood cells

Metabolic activation:

with and without

Genotoxicity:

other: DNA damage: positive for Tin II

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: human white blood cells

Metabolic activation:

without

Genotoxicity:

other: DNA damage: negative for Tin IV

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

without

Genotoxicity:

other: DNA damage: positive for Tin II

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: white blood cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative For Tin IV
positive For Tin II

This report documents the potential of tin(II), but not tin(IV), to enter human WBC and cause DNA damage.

Executive summary:

To provide information about possible genotoxic effects in man, the DNA damaging effects of in vitro exposure of human white blood cells to tin compounds was studied. Meanwhile the uptake capability of Tin II and Tin IV were also evaluated by human white blood cells (WBS). In addition, the DNA strand break damage of WBS after exposure to Tin II and Tin IV was also examined by fluorometric analysis of DNA unwinding (FADU). In the report it also evaluate the further effects of Tin II and Tin IV on Con A-stimulated DNA synthesis in peripheral blood lymphocytes HPBL and mouse splenocytes. From the test, the damage was detected in WBC after exposure of 5-50μM tin(II) for 30 min at both 0 and 37°C. Additionally, pretreatment of human lymphocytes with tin(II) prior to Con A stimulation was also found to inhibit the rate at which [3H]TdR was incorporated into DNA. This report documents the potential of tin(II), but not tin(IV), to enter human WBC and cause DNA damage. An apparent threshold for damage was observed, which in part, reflects a ,threshold for the uptake of tin(II) by cells in an in vitro system. However, The rate of DNA synthesis was not impaired in cells exposed to tin(IV).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Additional information

The normal body mechanisms governing absorption, excretion, retention, and turnover of tin suggest a low toxicological risk from the ingestion of tin salts. Various animal data on inorganic compounds indicate even lower toxicity of Tin (IV) than Tin (II) and insoluble inorganic tin than soluble inorganic tin. Both Tin (II) oxide and Tin (IV) dioxide didn’t induce any effect in rats in repeated dose toxicity studies.

Based on the above, the use of Tin (IV) chloride as a structural surrogate for Tin (IV) dioxide (CAS No. 18282-26-4) for Mutagenicity studies is feasible.

Several tin compounds have failed to give evidence of an ability to induce DNA damage in Bacillus subtilis , as assessed by the relative survival of wild-type and DNA repair-deficient strains (rec assays) on exposure to tin. Using B. subtilis strains H17(rec+) and H45(rec–), four tin salts (tin(II) chloride, tin(IV) chloride, tin(II) sulphate, and sodium stannate) gave no evidence of an ability to cause DNA damage, although the investigators noted that the two chlorides were highly toxic to the bacteria, which would have reduced the sensitivity of the test (Kada et al., 1980). Tin(II) chloride, tin(IV) chloride, and sodium stannate were similarly inactive in this assay in the absence of any added metabolic activation fraction (Nishioka, 1975). The literature contains another report of a rec assay test with B. subtilis in which tin(IV) chloride, when tested at up to 10 mg, gave no evidence of an ability to damage DNA (Hamasaki et al., 1992). In the same research, Tin(IV) chloride was determined that it cannot produce DNA damage in an SOS chromotest (Hamasaki et al., 1992). In addition, Tin(IV) chloride gave no evidence of mutagenic potential in a more limited study using only S. typhimurium strains TA98 and TA100 (Hamasaki et al., 1993).

Tin (II) chloride at concentrations of 50, 150, 350, or 500µmol/litre produced dose-related DNA damage, as detected by alkaline sucrose gradient analysis in Chinese hamster ovary cells. Treatment of cells with tin (IV) as tin (IV) chloride produced no such DNA damage. There was no loss in colony formation 6 days after either treatment (McLean et al., 1983a). In a further study, Tin(II) as tin(II) chloride (5, 10, 25, or 50µmol/litre) was readily taken up by human white blood cells and caused a dose-dependent increase in DNA strand breaks that was more extensive than that caused by equimolar amounts of chromium(VI), a known carcinogen and DNA-damaging agent. Tin (IV) as tin (IV) chloride did not cause DNA damage and, in contrast to other studies, was not taken up by cells (McLean et al., 1983b). According to a report published, incubation of human lymphocytes from 27 male donors with tin (IV) chloride at 2 or 4 µg/ml for 70 h resulted in 2- to 3-fold increases in the incidences of chromosome aberrations and SCEs (Ganguly et al., 1992). However the extremely high dose of stannic chloride (20 µg/ 5 ml culture) was used. The significantly higher clastogenicity of stannic chloride may be related to the much large amounts of the salt used and to the high frequency of inter-individual variation. Inter- and intra-individual variations are important factors in assessing chromosomal damage (Obe and Beek, 1984) and variations in chromosomal aberrations and sister-chromatid exchanges in control populations are well known (Dewdney et al., 1986; Ghosh et al., 1988b; Obeet al., 1984). Therefore the highly increased incidences of chromosome aberrations and SCEs may be due to the much large amounts of the salt used but not to the Tin IV per se.

Any additional in vitro mammalian cell gene mutation assay has been performed as, based on the knowledge of the substance, it would not provide further useful information about the in vivo mutagenicity of the substance (refer to ECHA “Guidance on information requirements and chemical safety assessment Chapter R.7a: Endpoint specific guidance”)

To conclude, tin (IV) chloride and related to tin dioxide have no potential for genotoxicity nor for cyto-genotoxicity.

Short description of key information:
1) Tin(IV) chloride has failed to give evidence of an ability to induce DNA damage in Bacillus subtilis, as assessed by the relative survival of wild-type and DNA repair-deficient strains (rec assays) on exposure to Tin(IV) chloride.
2) Among 144 metal compounds rec-assayed with cold incubation, SnCl2 and SnCl4 both resulted to be negative for DNA damage.
3) SnCl4 doesn't show a significant increase of His+ revertants in the assays with both the type of S typhimurium strain TA 100 and TA 98.
4) It indicates that tin(II) can produce extensive DNA damage in CHO cells without affecting survival, but not for Tin IV.
5) It indicated Tin(IV) chloride did not produce DNA damage in an SOS chromotest.
6) Extremely high dose of stannic chloride can induce a much high frequency of chromosomal abnormalities and a great reduction of cell cycle kinetics.
7) It documented the potential of tin(II), but not tin(IV), to enter human WBC and cause DNA damage.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on existing information, the substance is not need to be classified under Regulation (EC) No 1272/2008 for any category of germ cell mutagenicity. For the same reason it does not satisfy with the classification criteria of Directive 67/548/eec.