Tin disulphide

Genetic toxicity: in vitro

Administrative data

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to standard methods and GLP and is considered adequate, reliable and relevant for classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tin disulphide

EC Number:

215-252-9

EC Name:

Tin disulphide

Cas Number:

1315-01-1

Molecular formula:

S2Sn

IUPAC Name:

tin disulphide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

hisitdine; Salmonella typhimurium
tryptophan; Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: nutrient agar
- Properly maintained: yes

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: nutrient agar
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

preliminary toxicity test: 50, 100, 200, 400, 800, 1600, 3200 and 5000 µg/plate (TA 100)
initial mutation assay: 50, 158, 500, 1580 and 5000 µg/plate (plate incorporation method)
confirmatory assay: 100, 266, 707, 1880 and 5000 µg/plate (preincubation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water (SW)
- Justification for choice of solvent/vehicle: a suspesion was made in sterile water by stirring for 20 minutes

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

100µL

Positive controls:

yes

Remarks:

Salmonella strains 4µg/plate; E. Coli 30µg/plate

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 98 , TA 100 , TA 1535, TA 1537, WP2uvrA (+S-9)

Controls 2

Positive controls:

yes

Remarks:

2 µg/plate

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 (-S-9)

Controls 3

Positive controls:

yes

Remarks:

50 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 (-S-9)

Controls 4

Positive controls:

yes

Remarks:

1 µg/plate

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 (-S-9)

Controls 5

Positive controls:

yes

Remarks:

4 µg/plate

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

WP2uvrA (-S-9)

Details on test system and experimental conditions:

Initial mutation assay:
METHOD OF APPLICATION: in agar (plate incorporation & preincubation)
DURATION
- Expression time (cells in growth medium): 67 hours
SELECTION AGENT (mutation assays):histidine/tryptophan
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method:number of revertants/plate

Confirmatory mutation assay:
METHOD OF APPLICATION:
- Preincubation period: approximately 30 minutes
- Expression time (cells in growth medium): 67 hours
SELECTION AGENT (mutation assays): histidine/tryptophan
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: number of revertants/plate

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for the strains TA98, TA100 and WP2uvrA (pKM 101) or equal to or greater than 3 times the mean vehicle control value for the strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Initial and confirmatory mutation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

slight precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Initial and confirmatory mutation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

slight precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Initial and confirmatory mutation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

slight precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Initial and confirmatory mutation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

slight precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

Initial and confirmatory mutation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

slight precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: distinguished by a healthy background lawn comparable to vehicle control plates

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1.a. Summary results of Initial Bacterial Reverse Mutation Assay in the Presence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98		TA100		TA1535		TA1537		WP2uvrA (pKM101)
	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b
Vehicle control (SW 100µL)	28	NA	105	NA	16	NA	11	NA	122	NA
50	26	0.94	111	1.06	16	1.04	10	0.94	132	1.08
158	28	1.00	120	1.15	16	1.04	9	0.85	143	1.17
500	27	0.96	111	1.06	14	0.89	10	0.94	133	1.09
1580	25	0.90	109	1.04	13	0.81	10	0.94	136	1.12
5000	25	0.90	116	1.11	14	0.89	10	0.88	128	1.05
Positive control	533^c	19.25^c	891^c	8.51^c	144^c	9.19^c	109^c	9.91^c	560^d	4.59^d
^aValues are means of three replicates and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4µg/plate) ^dWP2uvrA (pKM101): 2-Aminoanthracene (30µg/plate) NA: Not applicable

 

Table 1.b. Summary results of Initial Bacterial Reverse Mutation Assay in the Absence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98		TA100		TA1535		TA1537		WP2uvrA (pKM101)
	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b
Vehicle control (SW 100µL)	26	NA	108	NA	14	NA	10	NA	124	NA
50	22	0.87	112	1.06	13	0.95	11	1.07	128	1.04
158	27	1.04	113	1.05	14	1.00	10	0.97	111	0.89
500	26	1.00	111	1.03	13	0.93	9	0.93	121	0.98
1580	23	0.88	109	1.01	14	1.02	7	0.73	128	1.04
5000	23	0.88	106	0.98	12	0.88	8	0.83	122	0.99
Positive control	251^c	9.78^c	558^d	5.16^d	154^d	11.24^d	120^e	12.03^e	548^f	4.43^f
^aValues are means of three replicates and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98: 2-Nitrofluorene (2µg/plate) ^dTA100, TA1535: Sodium azide (1µg/plate) ^eTA1537: 9-Aminoacridine (50µg/plate) ^fWP2uvrA (pKM101): 4-Nitroquinoline-1-oxide (4µg/plate) NA: Not applicable

 

Table 2.a. Summary results of Confirmatory Bacterial Reverse Mutation Assay in the Presence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98		TA100		TA1535		TA1537		WP2uvrA (pKM101)
	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b
Vehicle control (SW 100µL)	25	NA	101	NA	14	NA	11	NA	126	NA
100	23	0.92	103	1.02	12	0.85	11	1.03	128	1.01
266	26	1.04	108	1.07	17	1.24	11	1.06	126	1.00
707	21	0.83	109	1.08	18	1.32	12	1.09	131	1.04
1880	22	0.86	107	1.06	14	1.00	11	1.00	126	1.00
5000	23	0.89	103	1.02	15	1.12	11	1.00	128	1.00
Positive control	525^c	20.74^c	870^c	8.61^c	142^c	10.39^c	105^c	9.81^c	554^d	4.39^d
^aValues are means of three replicates and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4µg/plate) ^dWP2uvrA (pKM101): 2-Aminoanthracene (30µg/plate) NA: Not applicable

 

Table 2.b. Summary results of Confirmatory Bacterial Reverse Mutation Assay in the Absence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98		TA100		TA1535		TA1537		WP2uvrA (pKM101)
	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b	Mean	Ratio^b
Vehicle control (SW 100µL)	24	NA	100	NA	14	NA	10	NA	129	NA
100	23	0.97	101	1.01	14	0.95	10	1.03	116	0.90
266	24	1.00	99	0.99	13	0.88	11	1.10	129	1.00
707	21	0.89	104	1.05	14	0.98	12	1.17	119	0.93
1880	22	0.94	107	1.07	14	0.98	10	1.00	123	0.95
5000	21	0.89	105	1.05	14	1.00	10	1.03	128	0.99
Positive control	235^c	9.92^c	544^d	5.45^d	145^d	10.14^d	110^e	11.00^e	548^f	4.25^f
^aValues are means of three replicates and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98: 2-Nitrofluorene (2µg/plate) ^dTA100, TA1535: Sodium azide (1µg/plate) ^eTA1537: 9-Aminoacridine (50µg/plate) ^fWP2uvrA (pKM101): 4-Nitroquinoline-1-oxide (4µg/plate) NA: Not applicable

 

Applicant's summary and conclusion

Conclusions:

The study indicated that Tin disulfide (CAS 1315-01-1) was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest tested dose of 5000 µg/plate.

Executive summary:

The test item, Tin disulfide (CAS 1315-01-1) was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli. The study consisted of a preliminary toxicity test and two mutation assays comprising four independent experiments. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver). Tin disulfide formed a suspension in sterile water (SW) at 50 mg/mL and was found to be homogeneous and stable up to 24 hours at 1 and 200 mg/mL when stored at room temperature. 
In a preliminary toxicity test, the mean number of revertant colonies was more or less comparable to the SW control plates up to the highest tested dose of 5000 µg/plate, both in the presence and absence of metabolic activation. No toxicity of the test item was seen as the intensity of the bacterial background lawn was comparable to that of the SW control plates up to 5000 µg/plate, both in the presence and absence of metabolic activation. There was a slight precipitation of the test item on the basal agar plates at 5000 µg/plate both in the presence and absence of metabolic activation. Based on these observations, it was decided to test up to the maximum dose of 5000 µg/plate in the initial as well as the confirmatory mutation assay.
In the initial mutation assay, the test item was exposed in triplicate to 50, 158, 500, 1580 and 5000 µg/plate test doses in the presence and absence of metabolic activation using direct plate incorporation procedure. In the confirmatory assay, the test item was exposed in triplicate to concentrations of 100, 266, 707, 1880 and 5000 µg/plate in the presence and absence of metabolic activation using pre-incubation procedure. The vehicle control (SW) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.
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 in the presence or absence of metabolic activation.The mean numbers of revertant colonies neither doubled for strains TA98, TA100 and WP2uvrA (pKM101) nor tripled for strains TA1535 and TA1537 when compared to the respective vehicle control plates, either in the presence or in the absence of the metabolic activation at any of the tested doses.
Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.
The study indicated that Tin disulfide (CAS 1315-01-1) was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest tested dose of 5000 µg/plate.