Tin sulfide (Sn2S3)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-08 to 2010-03-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study on a structural analogous read-across substance (Please refer to the Read-Across Justification in Section 13)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

, published in O.J L 142, 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

, adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tin sulphide

EC Number:

215-248-7

EC Name:

Tin sulphide

Cas Number:

1314-95-0

Molecular formula:

SSn

IUPAC Name:

Tin(II) sulfide

Constituent 2

Reference substance name:

Tin sulfide

IUPAC Name:

Tin sulfide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

The bacterial tester strains · histidine dependent Salmonella typhimurium TA 98 (CCM 3811), TA 1535 (CCM 3814), and tryptophan dependent strain Escherichia coli WP2 uvrA (CCM 4751) - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno and TA100 (CIP 103796, lot. No.1008) and TA 1537 (CIP 103799, lot No. 34508) were from Biological Resource Center of Institut Pasteur (CRBIP), Paris Strains TA 1537 and TA 98 detect frame shift mutations, strains TA 100 and TA 1535 serve to detect base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The test substance was suspended in water for injections and assayed in doses of 50-5000 µg which were applied to plates in volume of 0.1 mL.
Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

Vehicle / solvent:

Suspension in water for injections, because the test substance is not soluble in any solvent
Preparation and using of S9: Delor 106 was diluted with olive oil

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylenediamine; 2-aminofluorene; 2-aminoanthracene; 9-aminoacridine hydrochloride monohydrate; N-methyl-N' -nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Test procedure: 100 µL of tin sulfide of required concentration, 0.1 mL 16-18 h culture of tester strain, 0.5 mL relevant buffer and 30 or 100 µL of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL top agar (with trace ofhistidine or tryptophan) kept in a test tube at 45± 3°C. After shaking the mixture was poured into a minimal glucose agar plate. After incubation of 48- 72 hat 37 ± 1 °C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000. For an adequate estimate of variation, triplicate plating was used at each dose level except in the toxicity test with strain TA 100, where test substance was tested in duplo. Each experiment was repeated. As there was no cytotoxicity, no precipitation or dose-responsiveness, doses in the second experiments remained the same as in the first experiment. In case of toxicity or precipitation, toxic (precipitating) doses would be omitted. In case of mutagenicity, such doses would be chosen which allow construction of a dose-response curve.
Selection of doses/toxicity As the test substance is not soluble in any solvent, a suspension in water for injections was prepared in maximum concentration given in guidelines (5000 µg per plate). Visual inspection was performed to confirm homogeneity of the suspension. The highest concentration was further diluted. Single test tubes were shaken before withdrawal of an aliquote for dilution as well as before withdrawal of 0.1 mL to top agar and before pouring of top agar to dishes. Concentration series arised (10-5000 µg per plate) was tested for toxicity in strain TA 98 without metabolic activation. Although the suspension of the test substance was present in top agar, the evaluation was possible without problems. No signs of toxicity were observed. The first mutagenicity experiments were done with the same highest dose as toxicity test. The starting dose was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. √10) intervals between test points). No problems occured at evaluation, so the same doses were used in the second mutagenicity experiments.
All concentrations of the test substance suspension were dosed in the volume of 0.1 mL per plate. Fresh suspensions of test substance were prepared before each experiment. The suspensions were shaken during dilution, before dosing to the top agar and before pouring onto the plates.
Preparation and using of S9: The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs ). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 wasprepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCL The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below -70 °C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer. Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphatc and 30 or 100 µL S9 (the concentration of S9 in the S9 mix was 5.7 or 16.6%). In experiments without metabolic activation only buffer was added to the top agar.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tin sulfide was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Tin sulfide is non mutagenic for all tested bacterial strains with and without metabolic activation.

Executive summary:

Tin sulfide was assayed for the mutagenicity by the Bacterial Reverse Mutation Test according to EU method B.13/14 Mutagenicity - Reverse mutation test using bacteria. Four indicators Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in water for injections and assayed in doses of 50-5000 µg which were applied to plates in volume of 0.1 mL. Tin sulfide was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.

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