Tin sulphide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tin sulfide was tested in following key in vitro genotoxicity assays:

-  Bacterial Reverse Mutation Assay using Salmonella typhimurium (TA98, TA100, TA1535 and TA 1537) and Escherichia coli (WP2 uvrA) was tested negative up to concentrations of 5000 µg/plate in presence and absence of metabolic activation (Plate incorporation and Preincubation).

-   In vitro mammalian chromosome aberration test in human lymphoblastoid cells (TK6) was tested negative up to 5.0 mg/mL with and without metabolic activation at predetermined intervals (4 and 26 hours).

-   In vitro mammalian gene mutation assay in Chinese hamster lung fibroblasts (V79) cells at the Hprt locus was tested negative up to 2000 µg/mL for a 4 hour exposure time in the absence and presence of metabolic activation.

These results were confirmed by in vitro studies on the read-across substance tin disulfide. The genotoxic potential of the read-across test item Tin Disulfide (CAS 1315-01-1) was also demonstrated not to induce bacterial or gene mutation in mammalian cells nor chromosome aberration in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2019 - 18 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 July 2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Council Regulation (EC) No. 440/2008 of 30 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Tribotecc, batch S903028
- Expiration date of the lot/batch: 31 March 2020
- Purity test date: 21 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed original container and stored in a dry place.
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: As Tin(II)-sulfide was not soluble in any of the solvents recommended the test item was suspended in highly purified water to a stable suspension of 200 mg/mL and diluted to the lower concentrations. Fresh preparations of the test item were used for the treatment in all experimental parts.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: As Tin(II)-sulfide was not soluble in any of the solvents recommended the test item was suspended in highly purified water to a stable suspension of 200 mg/mL and diluted to the lower concentrations. Fresh preparations of the test item were used for the treatment in all experimental parts.


OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: The following pH data of the negative control and of the test item formulations in the medium were determined in the dose-range-finding study:
Medium : pH 7.50
Negative control: pH 7.45
3.16 µg/mL Tin (II)-sulfide: pH 7.55
10.0 µg/mL Tin (II)-sulfide: pH 7.53
31.6 µg/mL Tin (II)-sulfide: pH 7.57
100 µg/mL Tin (II)-sulfide: pH 7.52
316 µg/mL Tin (II)-sulfide: pH 7.55
1000 µg/mL Tin (II)-sulfide: pH 7.57
2000 µg/mL Tin (II)-sulfide: pH 7.57
The following osmolality data of the negative control and of the test item formulations in the medium were determined in the dose-range-finding study:
Medium : 325 mOsmol/kg
Negative control: 305 mOsmol/kg
3.16 µg/mL Tin (II)-sulfide: 310 mOsmol/kg
10.0 µg/mL Tin (II)-sulfide: 310 mOsmol/kg
31.6 µg/mL Tin (II)-sulfide: 310 mOsmol/kg
100 µg/mL Tin (II)-sulfide: 315 mOsmol/kg
316 µg/mL Tin (II)-sulfide: 305 mOsmol/kg
1000 µg/mL Tin (II)-sulfide: 308 mOsmol/kg
2000 µg/mL Tin (II)-sulfide: 305 mOsmol/kg

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cells, Lot 4; DSMZ, Braunschweig, Germany
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: The cells were periodically checked for the absence of mycoplasma contamination.
- Number of passages if applicable: 14th or 16th passage
- Methods for maintenance in cell culture: V79 cells were maintained in growth medium Dulbecco's modified Eagle-Medium (DMEM) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin solution (14th or 16th passage). Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2. For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: The spontaneous mutation rate was continuously monitored.


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
*growth medium: Dulbecco's modified Eagle-Mediu (DMEM) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin solution. Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2.
*For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
*treatment medium: Dulbecco's modified Eagle-Medium) supplemented with 5% foetal calf serum and 1% penicillin/streptomycin solution.

For experiments without metabolic activation 0.15 mL test item solution, negative or positive controls were added to 14.85 mL treatment medium.
For experiments with metabolic activation 3 mL of the S9 mix were added to 11.85 mL treatment medium before treatment.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem
- method of preparation of S9 mix: Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 (Monsanto KL615, 500 mg/kg i.p.) and prepared according to MARON and AMES was obtained from Trinova Biochem . S9 was collected from male rats. The S9 fraction was stored at below -80°C until use.
MARON, D. M. and B. N. AMES. Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173 - 215 (1983).
- concentration or volume of S9 mix and S9 in the final culture medium :
The S9 fraction was thawed immediately before use and was combined to form the activation system described below:
270.0 mg glucose-6-phosphate
37.5 mg NADP
3.0 mL 150 mM KCl salt solution (sterile stock solution)
3.0 mL rat liver S9 (Aroclor 1254-induced)
24 mL Dulbecco's Phosphate-Buffered Saline (DPBS)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The protein content of the S9 fraction and the cytochrome P-450 activity was tested by Moltox and distributed by Trinova Biochem. The S9 mix was sterilised through a 0.45 µm filter and kept on ice at all times. The S9 mix was always prepared freshly for each experiment.

Test concentrations with justification for top dose:

The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium. In this preliminary test no signs of cytotoxicity in form of decreased relative survival compared to the control were noted up to the top concentration of 2000 µg Tin(II)-sulfide/mL medium in the absence and presence of metabolic activation. Test item precipitation was noted macroscopically at all concentrations in both experiments. No relevant changes in pH or osmolality were noted in the test cultures compared to the negative control treated with highly purified water. Hence, 2000 µg Tin(II)-sulfide/mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation.

Main test: Concentrations of 125, 250, 500, 1000 and 2000 μg Tin(II)-sulfide/mL medium were selected for the mutagenicity experiments without or with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: highly purified water

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

600 and 700 µg EMS/mL medium ; 20 and 30 µg DMBA/mL medium

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
>For determination of cytotoxicity (relative plating efficiency, PE1): 3 replicate plates
>For determination of the mutant frequency :
*selection of mutants: 4 replicate plates
*estimation of plating efficiencies (PE2): 3 replicate plates
- Number of independent experiments : 2 experiments with and 2 experiments without S9 mix

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Approximately 1 500 000 cells were placed in 15 mL DMEM-FCS per 75 cm2 culture flask.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No
- Exposure duration/duration of treatment: 4 hours treatment (with or without S9)
- Harvest time after the end of treatment (sampling/recovery times):
CYTOTOXICITY: 8 days for determination of relative plating efficiency (PE1)
MUTAGENICITY: after 6 days (including 1 cell passage in between) expression time
*about 8 days fot the estimation of plating efficiencies (PE2)
*about 12 days (mutant selection)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 6 days (including 1 cell passage in between)
- Selection time (if incubation with a selective agent): about 12 days (with 6-thioguanine)
- Fixation time (start of exposure up to fixation or harvest of cells):
CYTOTOXICITY: 4 hours treatment + 8 days for determination PE1
MUTAGENICITY:
4 hours treatment + 6 days expression time + about 8 days for determination PE2
4 hours treatment + 6 days expression time + about 12 days for mutant selection
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 6-thioguanine 10 µg/mL for about 12 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: other: relative plating efficiency; relative survival (RS)
- Any supplementary information relevant to cytotoxicity: At the end of the exposure period and removal of the test item the cells were washed with PBS and the cells were trypsinised and then suspended in 9 mL growth medium. The cells were pelleted by centrifugation (250 x g for 5 minutes), the supernatant was removed, and the cells were resuspended in 3 mL growth medium.
Then, one part of the cells was used for the determination of the relative plating efficiency for each dose to obtain an accurate measure of the cytotoxic effect of the chemical. Therefore, three replicate plates (60 mm diameter dishes) were used with 150 cells per plate in 5 mL growth medium. After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted for plating efficiency (PE1). Relative Survival were calculated using the following equations:
Relative Survival (RS) [%] = [PE1 (treated culture) / PE1 (control culture)] x 100.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
At the end of the exposure period and removal of the test item the cells were washed with PBS and the cells were trypsinised and then suspended in 9 mL growth medium. The cells were pelleted by centrifugation (250 x g for 5 minutes), the supernatant was removed, and the cells were resuspended in 3 mL growth medium.
A part of the cells was used for the determination of the mutant frequency. The cells were further incubated for 6 days including one cell passage in between. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously expressed HPRT. After the expression period the cells were harvested by trypsinisation and replated at a density of 500 000 cells per 100 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants (4 replicate plates), and 150 cells per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates). After about 8 days (PE2) or about 12 days (mutant plates), the cells were fixed and stained with methylene blue in ethanol and the colonies were then counted.

- OTHER: The pH and osmolality of the negative control and all test item formulations in the medium were determined employing the methods given below:
pH values: using a digital pH meter type SevenCompact s’210 (Mettler-Toledo GmbH, 35396 Gießen).
Osmolality: with a semi-micro osmometer13F( KNAUER, 14163 Berlin, Germany).

Evaluation criteria:

Acceptance of a test is based on the following criteria:
-The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
-Concurrent positive controls induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
-Four tested concentrations are analysable.
The spontaneous mutation frequency may vary from experiment to experiment, but should normally lie within the range of background data obtained at LPT.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test item precipitation was noted macroscopically at all concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 44% relative survival compared to the control was noted in the second experiment without metabolic activation at the top concentration of 2000 µg Tin(II)-sulfide/mL medium

Remarks:

. No signs of cytotoxicity were noted up to the top concentration of 2000 µg Tin(II)-sulfide/mL medium in the first experiment without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The following pH data of the negative control and of the test item formulations in the medium were determined in the dose-range-finding study:
Medium : pH 7.50
Negative control: pH 7.45
3.16 µg/mL Tin (II)-sulfide: pH 7.55
10.0 µg/mL Tin (II)-sulfide: pH 7.53
31.6 µg/mL Tin (II)-sulfide: pH 7.57
100 µg/mL Tin (II)-sulfide: pH 7.52
316 µg/mL Tin (II)-sulfide: pH 7.55
1000 µg/mL Tin (II)-sulfide: pH 7.57
2000 µg/mL Tin (II)-sulfide: pH 7.57
- Data on osmolality: The following osmolality data of the negative control and of the test item formulations in the medium were determined in the dose-range-finding study:
Medium : 325 mOsmol/kg
Negative control: 305 mOsmol/kg
3.16 µg/mL Tin (II)-sulfide: 310 mOsmol/kg
10.0 µg/mL Tin (II)-sulfide: 310 mOsmol/kg
31.6 µg/mL Tin (II)-sulfide: 310 mOsmol/kg
100 µg/mL Tin (II)-sulfide: 315 mOsmol/kg
316 µg/mL Tin (II)-sulfide: 305 mOsmol/kg
1000 µg/mL Tin (II)-sulfide: 308 mOsmol/kg
2000 µg/mL Tin (II)-sulfide: 305 mOsmol/kg

RANGE-FINDING/SCREENING STUDIES (if applicable): To determine cytotoxicity, the same procedure was used in the preliminary cytotoxicity test as employed for the mutagenicity experiments except that no mutant selection was carried out. Approximately 1 500 000 cells were placed in 15 mL growth medium per 75 cm2 culture flask. On the following day, the growth medium was removed and the cells were resuspended in treatment medium (Dulbecco's modified Eagle-Medium) supplemented with 5% foetal calf serum and 1% penicillin/streptomycin solution. The cells were exposed to a wide range of concentrations of Tin(II)-sulfide in the absence and presence of S9 mix, for 4 hours. For experiments without metabolic activation 0.15 mL test item solution, negative or positive controls were added to 14.85 mL treatment medium.
For experiments with metabolic activation 3 mL of the S9 mix were added to 11.85 mL treatment medium before treatment.
After removal of the test item and washing of the plates with PBS the cells were trypsinised and then suspended in 9 mL growth medium. The cells were pelleted by centrifugation (250 x g for 5 minutes), the supernatant was removed, and the cells were resuspended in 3 mL growth medium. Then the relative plating efficiency was determined for each dose to obtain an accurate measure of the cytotoxic effect of the chemical.
Three replicate plates (60 mm diameter dishes) were used with 150 cells in 5 mL growth medium. After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted for plating efficiency (PE). The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium. In this preliminary test no signs of cytotoxicity (i.e. ≤ 50% decrease of relative survival) compared to the control were noted in form of decreased relative survival up to the top concentration of 2000 µg Tin(II)-sulfide/mL medium in the absence and presence of metabolic activation but only 67% to 72% relative survival at concentrations of 1000 and 2000 µg Tin(II)-sulfide/mL. Test item precipitation was noted macroscopically at all concentrations in both experiments (see Table 1). No relevant changes in pH or osmolality were noted in the test cultures compared to the negative control treated with highly purified water. Hence, 2000 µg Tin(II)-sulfide/mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
*Mutagenicity Experiments without metabolic activation:
The mutation frequency of the solvent control highly purified water was 6.36 and 8.42 mutant colonies per 1 000 000 cells, for the 1st and the 2nd experiment, respectively. The positive control EMS (ethyl methanesulfonate) caused a pronounced increase in the mutation frequencies ranging from 222.94 to 651.00 mutant colonies per 1 000 000 cells in the case of EMS, indicating the validity of this test system.
*Mutagenicity Experiments with metabolic activation:
The mutation frequency of the solvent control highly purified water was 5.69 and 6.20 mutant colonies per 1 000 000 cells, for the 1st and the 2nd experiment, respectively.
The positive control DMBA (9,10- dimethyl-1,2-benzanthracene) caused a pronounced increase in the mutation frequencies ranging from 162.87 to 303.65 mutant colonies per 1 000 000 cells in the case of DMBA, indicating the validity of this test system.

Gene mutation tests in mammalian cells:
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
Approximately 1 500 000 cells were placed in 15 mL DMEM-FCS per 75 cm2 culture flask. On the following day, the growth medium was removed and the cells were resuspended in treatment medium. The cells were exposed to a wide range of concentrations of Tin(II)-sulfide in the absence and presence of S9 mix, for 4 hours.
For experiments without metabolic activation 0.15 mL test item solution, negative or positive controls were added to 14.85 mL treatment medium.
For experiments with metabolic activation 3 mL of the S9 mix were added to 11.85 mL treatment medium before treatment.
o Number of cells plated in selective and non-selective medium
At the end of the exposure period and removal of the test item the cells were washed with PBS and the cells were trypsinised and then suspended in 9 mL growth medium.
The cells were pelleted by centrifugation (250 x g for 5 minutes), the supernatant was removed, and the cells were resuspended in 3 mL growth medium.
Then, one part of the cells was used for the determination of the relative plating efficiency for each dose to obtain an accurate measure of the cytotoxic effect of the chemical. Therefore, three replicate plates (60 mm diameter dishes) were used with 150 cells per plate in 5 mL growth medium. After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted for plating efficiency (PE1).
Another part of the cells was used for the determination of the mutant frequency. The cells were further incubated for 6 days including one cell passage in between. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously expressed HPRT. After the expression period the cells were harvested by trypsinisation and replated at a density of 500 000 cells per 100 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants (4 replicate plates), and 150 cells per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates). After about 8 days (PE2) or about 12 days (mutant plates), the cells were fixed and stained with methylene blue in ethanol and the colonies were then counted.

HISTORICAL CONTROL DATA See under Any other information on material and methods incl. tables

Table 1                                   Preliminary cytotoxicity test

 

Culture number	Concentration of Tin(II)-sulfide		Plating Efficiency	Relative Survival
	[µg/mL]		PE1	RS [%]
without metabolic activation (4-hour exposure)
1	0	(control)	0.55	100
8		3.16#	0.62	112
7		10.0#	0.54	99
6		31.6#	0.58	105
5		100#	0.61	111
4		316#	0.62	112
3		1000#	0.40	72
2		2000#	0.37	67
with metabolic activation (4-hour exposure)
9	0	(control)	0.60	100
16		3.16#	0.60	99
15		10.0#	0.58	96
14		31.6#	0.61	101
13		100#	0.62	102
12		316#	0.54	90
11		1000#	0.63	104
10		2000#	0.66	109

# = test item precipitation

 

Table 2                 1. Experiment without metabolic activation (4-hour exposure)

 

Culture number	Concentration of Tin(II)-sulfide	Plating Efficiencies		Relative Survival	Thioguanine- resistant colonies			Cloning Efficiencies of mutant colonies			Mutation frequency
[µg/mL]		PE1	PE2	RS [%]	CE x					10-6	MF x	10-6
1	0 (control)	0.63	0.63	100	2	1	1	4	4.0		6.36
6	125#	0.55	0.66	87	3	2	3	1	4.5		6.86
5	250#	0.58	0.56	93	1	1	2	3	3.5		6.20
4	500#	0.44	0.53	70	1	1	1	1	2.0		3.78
3	1000#	0.59	0.57	94	2	2	3	2	4.5		7.85
2	2000#	0.48	0.56	75	2	2	1	2	3.5		6.20
7	EMS 600	0.56	0.48	89	60	41	62	53	108.0		222.94
8	EMS 700	0.46	0.50	73	151	140	168	192	325.5		651.00

# = test item precipitation

EMS = ethyl methanesulfonate

 

Table 3                 2. Experiment without metabolic activation (4-hour exposure)

 

Culture number	Concentration of Tin(II)-sulfide	Plating Efficiencies		Relative Survival	Thioguanine- resistant colonies			Cloning Efficiencies of mutant colonies			Mutation frequency
[µg/mL]		PE1	PE2	RS [%]	CE x					10-6	MF x	10-6
1	0 (control)	0.57	0.65	100	3	4	1	3	5.5		8.42
6	125#	0.50	0.52	88	1	3	2	1	3.5		6.79
5	250#	0.66	0.56	116	3	2	1	4	5.0		8.86
4	500#	0.47	0.53	82	2	1	1	0	2.0		3.78
3	1000#	0.41	0.63	72	3	2	2	1	4.0		6.38
2	2000#	0.25	0.63	44	1	0	1	3	2.5		3.99
7	EMS 600	0.52	0.46	91	78	75	85	72	155.0		338.6
8	EMS 700	0.51	0.41	89	102	104	96	96	199.0		486.7

 

 

# = test item precipitation

EMS = ethyl methanesulfonate

 

Table 4  1. Experiment with metabolic activation (4-hour exposure)

Culture number	Concentration of Tin(II)-sulfide	Plating Efficiencies		Relative Survival	Thioguanine- resistant colonies			Cloning Efficiencies of mutant colonies			Mutation frequency
[µg/mL]		PE1	PE2	RS [%]	CE x					10-6	MF x	10-6
9	0 (control)	0.59	0.62	100	1	1	3	2	3.5		5.69
14	125#	0.62	0.66	104	1	1	1	0	1.5		2.27
13	250#	0.47	0.63	79	2	1	1	0	2.0		3.19
12	500#	0.65	0.66	110	1	1	0	1	1.5		2.28
11	1000#	0.54	0.61	91	1	1	3	0	2.5		4.12
10	2000#	0.41	0.74	69	1	0	1	1	1.5		2.03
15	DMBA 20	0.21	0.50	35	48	54	49	50	100.5		202.80
16	DMBA 30	0.26	0.43	44	67	60	54	77	129.0		300.78

 

# = test item precipitation

EMS = ethyl methanesulfonate

 

 

Table 5                 2. Experiment with metabolic activation (4-hour exposure)

 

Culture number	Concentration of Tin(II)-sulfide	Plating Efficiencies		Relative Survival	Thioguanine- resistant colonies			Cloning Efficiencies of mutant colonies			Mutation frequency
[µg/mL]		PE1	PE2	RS [%]	CE x					10-6	MF x	10-6
9	0 (control)	0.60	0.56	100	2	3	0	2	3.5		6.20
14	125#	0.48	0.56	81	2	0	1	1	2.0		3.57
13	250#	0.59	0.50	99	0	1	0	3	2.0		4.02
12	500#	0.45	0.62	75	2	0	1	1	2.0		3.24
11	1000#	0.52	0.63	87	1	0	2	3	3.0		4.79
10	2000#	0.54	0.58	91	2	0	3	3	4.0		6.90
15	DMBA 20	0.27	0.53	45	49	36	50	38	86.5		162.87
16	DMBA 30	0.20	0.50	33	81	64	68	92	152.5		303.65

 

# = test item precipitation

EMS = ethyl methanesulfonate

Conclusions:

Under the present test conditions, Tin(II)-sulfide tested up to the concentration of 2000 µg/mL medium, in the absence and in the presence of metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

Tin(II)-sulfide was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.

As Tin(II)-sulfide was not soluble in any of the solvents recommended, the test item was suspended in highly purified water to a stable suspension of 200 mg/mL and diluted to the lower concentrations. The vehicle highly purified water was employed as the negative control.

Preliminary cytotoxicity test

The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg/mL medium. In this preliminary test no signs of cytotoxicity in form of decreased relative survival compared to the control were noted up to the top concentration of 2000 μg Tin(II)-sulfide/mL medium in the absence and presence of metabolic activation. Test item precipitation was noted macroscopically at all concentrations in both experiments. No relevant changes in pH or osmolality were noted in the test cultures compared to the negative control treated with highly purified water. Hence, 2000 μg Tin(II)-sulfide/mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation.

Main study

Concentrations of 125, 250, 500, 1000 and 2000 μg Tin(II)-sulfide/mL medium were selected for the mutagenicity experiments without or with metabolic activation. The experiments without and with metabolic activation were conducted in duplicates.

Cytotoxicity

Cytotoxicity in form of decreased relative survival (i.e. 44% relative survival) compared to the control was noted in the second experiment without metabolic activation at the top concentration of 2000 μg Tin(II)-sulfide/mL medium. No signs of cytotoxicity were noted up to the top concentration of 2000 μg Tin(II)-sulfide/mL medium in the first experiment without and both experiments with metabolic activation. Test item precipitation was noted macroscopically at all concentrations in both experiments each carried out without and with metabolic activation.

Mutagenicity

Experiments without metabolic activation

The mutation frequency of the solvent control highly purified water was 6.36 and 8.42 mutant colonies per 10⁶ cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 125, 250, 500, 1000 and 2000 µg Tin(II)-sulfide/mL culture medium ranged from 3.78 to 8.86 mutant colonies per 10⁶ cells. These results are within the normal range of the solvent controls.

Experiments with metabolic activation

The mutation frequency of the solvent control highly purified water was 5.69 and 6.20 mutant colonies per 10⁶ cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 125, 250, 500, 1000 and 2000 µg Tin(II)-sulfide/mL culture medium ranged from 2.03 to 6.90 mutant colonies per 10⁶ cells. These results are within the normal range of the solvent controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 222.94 to 651.00 mutant colonies per 10⁶ cells in the case of EMS and ranging from 162.87 to 303.65 mutant colonies per 10⁶ cells in the case of DMBA, indicating the validity of this test system.

Conclusion

Under the present test conditions, Tin(II)-sulfide tested up to the concentration of 2000 µg/mL medium, in the absence and in the presence of metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-08 to 2010-03-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

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

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

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 / 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

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 of histidine 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.

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

E. coli WP2 uvr A

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

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-14 to 2010-03-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

Cells: Human Peripheral Blood Lymphocytes (25mL whole blood treated with heparin)
Donor: Healthy female volunteer
Lymphocytes mitogen: Phytohaemagglutinin
Normal cell cycle time: 16-20h
Modal chromosome number: 46 chromosomes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary test: 0.05, 0.1, 0.2, 0.5, 1.0, 3.0, and 5.0 mg/mL in 1 mL of culture.
Main test: 1.0, 2.5, and 5.0 mg/mL in 1 mL of culture.
Main test No. 2: 1.0, 2.5, and 5.0 mg/mL in 1 mL of culture.
Repeated test: 1.0, 2.5, and 5.0 mg/mL in 1 mL of culture.

Vehicle / solvent:

-Vehicle/solvent used: 0.5 % methylcellulose

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Thiotepa

Details on test system and experimental conditions:

Cultivation medium was prepared in amount of 7.5 mL per culture tube, 0.5 mL of blood was added to cultivation medium and incubated at 37°C. Test item was added after 48 hours of cultivation.
The cultivation medium was prepared per 1 culture (without antibiotics) from:
RPMI 1640 5.34 mL
Fetal bovine serum 1.80 mL
Glutamine 0.10 mL
NaHC03 7.5% 0.16 mL
PHA HA 15 0.10 mL
Cells were exposed to the Test item and negative/positive controls both with and without MAS, and sampled at a time equivalent to about 1.5 normal cell cycle length (26 hours). At predetermined intervals (4 and 26 hours) after exposure of cell cultures to the Test item, they were treated with a metaphase-arresting substance - colchicine, harvested, and stained. Cell cultures were treated with colchicine for 2 hours prior to harvesting. Harvesting of cell cultures:
1. Centrifugation 3 min at 2000 rpm and discard the supernatant
2. Add 9.5 mL of hypotonic solution (0.55% KCl), incubate 10 min at room temperature
3. Centrifugation 3 min at 2000 rpm and discard the supernatant
4. Add 4.5 mL of acetic methanol with water
5. Centrifugation 3 min at 2000 rpm and discard the supernatant
6. Add 4.5 mL of methanol
7. Centrifugation 3 min at 2000 rpm and discard the supernatant
8. Add 4.5 mL of acetic methanol
9. Centrifugation 3 min at 2000 rpm and discard the supernatant
10. Resuspend and drop suspension on to slides
Slides were stained with Giemsa. Metaphase cells were analyzed microscopically for the presence of chromosome aberrations. In case of 4 hours exposition with metabolic activation the washing of cells was performed after exposition and cultivation continued for 26 hours using fresh medium.

Evaluation criteria:

Preliminary test:
The number of mitosis per 1000 cells was recorded. From these data the mitotic index was determined as a measure of cytotoxicity.
Main test:
200 well-spread metaphases with 46±2 centromeres were examined per concentration on coded slides
Main test No. 2 and Repeated test:
Four categories of chromosomal aberrations were evaluated: chromatid (Bl) and chromosome (B2) breaks and chromatid (El) and chromosome
(E2) exchanges. Chromosome exchange aberrations were detected by manual checking. Chromosome exchange aberrations were classified as dicentrics, centric rings or translocations. Chromatid gaps were not scored as aberrations. There number of breaks per cell (B/C) were calculated. Rules of calculating breaks per cell (B/C) is Bl, B2 = 1 break, El, E2 = 2 breaks. Then a frequency of aberrant cells (%AB) was calculated as a relationship of cells with chromosomal aberrations and 100 cells per cell culture.

The result are considered positive if the Test item increases the average % frequency of aberrant cells to more than twice that of the negative control value. The values of the historical negative controls are among 0-5 %of abenant cells. The criteria for positive results must also include the two-fold rule (increase in the number of chromosomal aberrations), the dose-response relationship and reproducibility of the results in the Repeated test.

Species / strain:

human lymphoblastoid cells (TK6)

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:

not examined

Positive controls validity:

valid

Additional information on results:

Tin sulfide did not induce an increase in numerical and structural chromosome aberrations in cultured peripheral blood lymphocytes.

Remarks on result:

other: strain/cell type: Lymphocytes mitogen: Phytohaemagglutinin

Conclusions:

Interpretation of results: negative

Tin sulfide does not induce numerical and structural chromosome aberrations in cultured mammalian somatic cells.

Executive summary:

The clastogenicity potential of tin sulfide was determined using In Vitro Chromosome Aberration Test according to OECD guideline 473. The test was carried out in human peripheral blood lymphocytes with and without in vitro metabolic activation system in two seperate assays. A concentration range of tin sulfide was used in the Preliminary test with and without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 3.0, and 5.0 mg/ml in 1 ml of culture. There were no cytotoxic effects both with and without metabolic activation. Three concentrations of the Test item: 1.0, 2.5, and 5.0 mg/ml in 1 ml of culture were used in the Main test. At predetermined intervals (4 and 26 hours) after exposure of cell cultures to the Test item, cells were arrested at metaphase with colchicine, harvested, and slides were stained. Metaphase cells were analyzed microscopically for the presence of chromosome aberrations. A total of 200 well-spread metaphases were examined per concentration on coded slides. Concurrent positive (Cyclophosphamide, Thiotepa) and negative (0.5% methylcellulose) controls were included in each experiment. Microscopic slides of the Main test could initially not be evaluated. Therefore a Main test No. 2 was performed with the same conditions as in the Main test. The microscopic slides related to the concentrations of 1.0, 2.5, and 5.0 mg/ml could be properly analyzed in the Main test No. 2 and in the Repeated test. There were no significant increases in the number of cells with chromosome aberrations and no trends of dose-response relationships. The negative results obtained were biologically relevant and reproducible. Under the test conditions used tin sulfide did not induce an increase in numerical and structural chromosome aberrations in cultured human peripheral blood lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Tin sulfide was tested in following key in vivo genotoxicity assays:

-   in vivo mammalian erythrocyte micronucleus test in male and female Wistar rats was negative after single exposure to 0, 500, 1000 and 2000 mg/kg bw via oral gavage.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-04 to 2010-03-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BioTest s. r. o., Pod Zámkem 279, Konárovice, Czech Republic
- Age at study initiation: 8-10 weeks
- Weight at study initiation: Males 250 g, Females 170 g
- Assigned to test groups randomly: yes, under following basis: sorted according to the body weight and randomly allocated to the dose group taking animals from each range group
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: methylcellulose 0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each animal in treated groups received the appropriate dose of Test item suspension orally by gavage in the volume of 1 mL per 100 g body weight. The animals in the negative control group received the appropriate volume of saline solution by the same way. The animals in the positive control group were administered cyclophosphamide solution at the dose of 20 mg/kg by a single intraperitoneal application in the volume of 1 mL per 100 g body weight.
The following procedure was adopted for the preparation of a homogenous suspension: at first the we ighed amount of the respective substance was ground in a grinding mortar with a small volume of carrier liquid. Then the content of the mortar was transferred into a calibrated vessel. Another amount of carrier liquid was poured into the mortar, stirred and the mortar with the pestle was rinsed, the rinsing liquid was added into the same calibrated vessel. This rinsing procedure was repeated once more. Then the volume in the calibrated vessel was made up with the carrier liquid to the required volume and mixed. Immediately before the application, the suspension was again stirred by means of an electromagnetic
stirrer.

Duration of treatment / exposure:

Duration of administration was 10-20 s

Frequency of treatment:

single exposure

Post exposure period:

One half of the animals from each group were sacrificed 24 hours after the administration and the other half of animals were sacrificed 48 hours after the administration.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

single exposure

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

single exposure

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

single exposure

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

single exposure

No. of animals per sex per dose:

Number of animals per group: 10 Males + 10 Females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

- Route of administration: i.p.
- Doses / concentrations: 20 mg/kg

Tissues and cell types examined:

bone marrow (mean number of NCPE, PCE, NCE and proportion of immature erythrocytes)

Details of tissue and slide preparation:

During the necropsy the bone marrow was extracted from femur using bovine serum into a labeled test tube. The cells were centrifuged (1000 rpm for 10 min). The supernatant was removed and the sediment was resuspended in approx. 4 drops of bovine serum. Smear preparations from this suspension were made in duplo for each animal and fixed with methanol for 5 min and then stained with Giemsa–Romanowski (1:10) for 15-20 min.
All the slides were independently coded before blind microscopic analysis.

Evaluation criteria:

At least 2000 polychromatic (immature) erythrocytes per animal were scored for the incidence of micronuclei under the microscope. The proportion of polychromatic (immature) among total (immature + mature or polychromatic + normochromatic) erythrocytes were determined for each animal by counting a total of at least 200 erythrocytes.

Statistics:

The analyzed data were tested for normality (Kolmogorov–Smirnov test) and homogeneity of variance (Bartlett’s test).
The data for males and females were separately analyzed using ANOVA followed by Dunnett’s Multiple Comparison Test. The F-test in the ANOVA tests whether there are any significant differences amongst the means at the 95.0 % confidence level.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tin sulphide does not produce micronuclei in the immature erythrocytes in the conditions of the test.

Conclusions:

Interpretation of results: negative
Tin sulfide does not produce micronuclei in the immature erythrocytes in the conditions of the test.

Executive summary:

The potential of tin sulfide to cause cytogenetic damage was assessed in a Mammalian Erythrocyte Micronucleus Test according to OECD guideline 474.

No changes in health status and condition of the animals in any of the groups were recorded during the acclimation and during the study. No significant changes of mean body weight were observed in the animals during the study. All the values of number of NPCE in all dose groups were within the reference range for negative control group. No statistically significant higher values of number of NPCE in any of the dose groups (up to 11 of micronuclei) as compared to negative control group (up to 13 of micronuclei) were noted. Statistically significant differences were observed in the positive control group (NPCE – up to 38 of micronuclei) as compared to control group.

Tin sulfide does not induce damage to the chromosomes or the mitotic apparatus of erythroblasts.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro and in vivo:

Ames test:

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 (Prochazkova, 2010). 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 non-mutagenic for all the used bacterial strains with as well as without metabolic activation.

Chromosomal Aberration Test (in vitro):

The clastogenicity potential of tin sulfide was determined using In Vitro Chromosome Aberration Test according to OECD guideline 473 (Kovarik, 2010). The test was carried out in human peripheral blood lymphocytes with and without in vitro metabolic activation system in two separate assays. A concentration range of tin sulfide was used in the Preliminary test with and without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 3.0, and 5.0 mg/mL in 1 mL of culture. There were no cytotoxic effects both with and without metabolic activation. Three concentrations of the Test item: 1.0, 2.5, and 5.0 mg/mL in 1 mL of culture were used in the Main test. At predetermined intervals (4 and 26 hours) after exposure of cell cultures to the Test item, cells were arrested at metaphase with colchicine, harvested, and slides were stained. Metaphase cells were analysed microscopically for the presence of chromosome aberrations. A total of 200 well-spread metaphases were examined per concentration on coded slides. Concurrent positive (Cyclophosphamide, Thiotepa) and negative (0.5% methylcellulose) controls were included in each experiment. Microscopic slides of the Main test could initially not be evaluated. Therefore a Main test No. 2 was performed with the same conditions as in the Main test. The microscopic slides related to the concentrations of 1.0, 2.5, and 5.0 mg/ml could be properly analysed in the Main test No. 2 and in the Repeated test. There were no significant increases in the number of cells with chromosome aberrations and no trends of dose-response relationships. The negative results obtained were biologically relevant and reproducible. Under the test conditions used tin sulfide did not induce an increase in numerical and structural chromosome aberrations in cultured human peripheral blood lymphocytes.

Mammalian gene mutation Test (in vitro):

Tin(II)-sulfide was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals (Spruth, 2019). The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix. As Tin(II)-sulfide was not soluble in any of the solvents recommended, the test item was suspended in highly purified water to a stable suspension of 200 mg/mL and diluted to the lower concentrations. The vehicle highly purified water was employed as the negative control.

The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg/mL medium. In this preliminary test no signs of cytotoxicity in form of decreased relative survival compared to the control were noted up to the top concentration of 2000 μg Tin(II)-sulfide/mL medium in the absence and presence of metabolic activation. Test item precipitation was noted macroscopically at all concentrations in both experiments. No relevant changes in pH or osmolality were noted in the test cultures compared to the negative control treated with highly purified water. Hence, 2000 μg Tin(II)-sulfide/mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation.

Concentrations of 125, 250, 500, 1000 and 2000 μg Tin(II)-sulfide/mL medium were selected for the mutagenicity experiments without or with metabolic activation. The experiments without and with metabolic activation were conducted in duplicates.

Cytotoxicity in form of decreased relative survival (i.e. 44% relative survival) compared to the control was noted in the second experiment without metabolic activation at the top concentration of 2000 μg Tin(II)-sulfide/mL medium. No signs of cytotoxicity were noted up to the top concentration of 2000 μg Tin(II)-sulfide/mL medium in the first experiment without and both experiments with metabolic activation. Test item precipitation was noted macroscopically at all concentrations in both experiments each carried out without and with metabolic activation.

Experiments without metabolic activation: The mutation frequency of the solvent control highly purified water was 6.36 and 8.42 mutant colonies per 106 cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range. The mutation frequency of the cultures treated with concentrations of 125, 250, 500, 1000 and 2000 µg Tin(II)-sulfide/mL culture medium ranged from 3.78 to 8.86 mutant colonies per 106 cells. These results are within the normal range of the solvent controls.

Experiments with metabolic activation: The mutation frequency of the solvent control highly purified water was 5.69 and 6.20 mutant colonies per 106 cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range. The mutation frequency of the cultures treated with concentrations of 125, 250, 500, 1000 and 2000 µg Tin(II)-sulfide/mL culture medium ranged from 2.03 to 6.90 mutant colonies per 106 cells. These results are within the normal range of the solvent controls. The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 222.94 to 651.00 mutant colonies per 106 cells in the case of EMS and ranging from 162.87 to 303.65 mutant colonies per 106 cells in the case of DMBA, indicating the validity of this test system.

Conclusion: Under the present test conditions, Tin(II)-sulfide tested up to the concentration of 2000 µg/mL medium, in the absence and in the presence of metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Mammalian Erythrocyte Micronucleus Test (in vivo):

The potential of tin sulfide to cause cytogenetic damage was assessed in a Mammalian Erythrocyte Micronucleus Test according to OECD guideline 474 (Prochazkova, 2010). No changes in health status and condition of the animals in any of the groups were recorded during the acclimation and during the study. No significant changes of mean body weight were observed in the animals during the study. All the values of number of NPCE in all dose groups were within the reference range for negative control group. No statistically significant higher values of number of NPCE in any of the dose groups (up to 11 of micronuclei) as compared to negative control group (up to 13 of micronuclei) were noted. Statistically significant differences were observed in the positive control group (NPCE – up to 38 of micronuclei) as compared to control group. Tin sulfide does not induce damage to the chromosomes or the mitotic apparatus of erythroblasts.

Read-across information from Tin Disulfide:(in vitro)

Ames test:

A key bacterial reverse mutation assay was performed with a suspension of Tin disulfide using TA98, TA100, TA1535 and TA1537 strains of S. typhimurium and WP2uvrA E. coli with and without metabolic activation (S9 fraction) prepared from Aroclor 1254 induced rat liver (Suresh, 2012). In a preliminary toxicity test, no cytotoxicity was seen up to 5000 µg/plate, with slight precipitation on the basal agar plates at 5000 µg/plate both with and without S9. Test doses were 50, 158, 500, 1580 and 5000 µg/plate using direct plate incorporation in the initial mutation assay and 100, 266, 707, 1880 and 5000 µg/plate using pre-incubation in the confirmatory assay. The results from the initial as well as from the confirmatory assays, indicate the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either with and without S9 up to the highest tested dose of 5000 µg/plate. A supporting study with read-across substance Tin sulfide using TA98, TA100, TA1535 and TA1537 strains of S. typhimurium and WP2uvrA E. coli with and without metabolic activation was also negative (Prochazkova, 2010). Tin disulfide and Tin sulfide showed a comparable toxicological profile for this endpoint.

 

Chromosome Aberration Test (in vitro):

A key study for chromosome aberrations was done with a suspension of Tin disulfide in cultured Chinese Hamster Ovary (CHO) cells, with and without metabolic activation (S9 fraction) prepared from Aroclor 1254 induced rat liver (Indrani, 2012). In a preliminary toxicity test, Tin disulfide showed evidence of significant growth inhibition at and above 229 µg/mL and 457 µg/mL during 3 hour exposure with and without S9, respectively, whereas in the absence of S9 with 21-hour exposure, there was evidence of significant reduction in the growth of CHO cells at and above 229 µg/mL. Exposure of Tin disulfide did not cause any appreciable change in the pH and osmolality of test solutions. In the definitive chromosome aberration assay, CHO cells were exposed to the test item in duplicate at concentrations of 23, 73 and 230 µg/mL (3 hours + S9 and 21 hours - S9) and at 30, 95 and 300 µg/mL (3 hours -S9). At the highest concentration tested at 3 hours (230 and 300 µg/mL), the reduction in the cell growth was 51% and 52% with and without S9, respectively, whereas at 21 hours without S9 (230 µg/mL), the reduction in cell growth was 53% when compared to the sterile water control. A total of 200 metaphases per dose level from duplicate cultures from the sterile water control, each treatment group and the positive control were evaluate for chromosome aberrations. There was no evidence of induction of chromosome aberrations, including or excluding gaps, either in the presence or in the absence of metabolic activation in any of these experiments. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases. As a supporting study, the clastogenicity potential of Tin sulfide was determined using In Vitro Chromosome Aberration Test (Kovarik, 2010). The test was carried out in human peripheral blood lymphocytes with and without metabolic activation system in two separate assays. Tin sulfide did not induce an increase in numerical and structural chromosome aberrations in cultured peripheral blood lymphocytes. Further, an in vivo micronucleus test according to OECD TG 474 was performed with Tin sulfide, also demonstrating absence of micronuclei in the immature erythrocytes in the bone marrow (Prochazkova, 2010). Tin disulfide and Tin sulfide showed a comparable toxicological profile for this endpoint.

Mammalian gene mutation (in vitro):

The genotoxic potential of the read-across test item Tin Disulfide (CAS 1315-01-1) to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells (Indrani, 2012). Both in the initial and confirmatory gene mutation assay, cells were exposed for 3 hours in the presence and absence of metabolic activation. There was no evidence of induction of gene mutations in any of the test material treated cultures either in the presence or absence of metabolic activation. The results of the forward gene mutation assay at the hprt locus indicated that there was no mutagenicity when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.

Justification for classification or non-classification

Based on the results obtained in in vitro and in vivo tests, tin sulfide is considered not to be genotoxic/mutagenic or clastogenic and thus had not to be classified according to Regulation (EC) No 1272/2008.