Tin sulphide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

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:

stannanethione

Test material form:

solid: particulate/powder

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

Method

Target gene:

hprt

Species / strain

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:

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Any other information on results 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

Applicant's summary and conclusion

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.