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Genetic toxicity: in vitro

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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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Target gene:
hisitdine; Salmonella typhimurium
tryptophan; Escherichia coli
Species / strainopen allclose all
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 / 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
Controlsopen allclose all
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)
Positive controls:
yes
Remarks:
2 µg/plate
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 (-S-9)
Positive controls:
yes
Remarks:
50 µg/plate
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (-S-9)
Positive controls:
yes
Remarks:
1 µg/plate
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535 (-S-9)
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
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
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/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

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

533c

19.25c

891c

8.51c

144c

9.19c

109c

9.91c

560d

4.59d

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/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

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

251c

9.78c

558d

5.16d

154d

11.24d

120e

12.03e

548f

4.43f

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/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

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

525c

20.74c

870c

8.61c

142c

10.39c

105c

9.81c

554d

4.39d

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/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

Mean

Ratiob

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

235c

9.92c

544d

5.45d

145d

10.14d

110e

11.00e

548f

4.25f

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.