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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:
11 February 2010 - 22 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification Sodium polysulfide solution
Chemical Name Sodium polysulfide
Molecular formula Na2Sx (x=2.48)
Molecular weight 125.498
CAS Number 1344-08-7
Description Reddish brown liquid
Batch 707-1
Purity TO BE ADDED!!
Test substance storage In refrigerator (2-8°C) in the dark under nitrogen
Stability under storage conditions Stable
Expiry date 03 June 2010

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Main study (without and with S9) TA1535, TA1537 and TA98: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.

Experiment 2:
Main study (without and with S9): 33, 100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: the test substance is 25% sodium polysulfide in water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: 60 µg/plate in milli-Q water for TA1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was observed.


Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) The increase in the mean number of revertant colonies follows the concentration of test substance (dose-response relationship).

Results and discussion

Test resultsopen allclose all
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
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- See below.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Toxicity:

Table 1

Reduction of the bacterial background lawn and in the number of revertant colonies

Strain

Without S9-mix

With S9-mix

                 Dose          Bacterial              Revertant

                (μg/plate)    background lawn  colonies

Dose       Bacterial                Revertant

(µg/plate) background lawn  colonies

TA1535

1000          slight                 -1

3330          moderate           -1

5000          extreme        microcolonies

3330          extreme        microcolonies

5000          extreme        microcolonies

TA1537

3330          extreme        microcolonies

5000          extreme        microcolonies

1000          slight                  -1

3330          extreme        microcolonies

5000          extreme        microcolonies

TA98

3330          extreme        microcolonies

5000          extreme        microcolonies

5000          -2                        -1

-1  No reduction in the number of revertant colonies

-2       No reduction of the bacterial background lawn

Table 2.

Reduction of the bacterial background lawn and in the number of revertant colonies

Strain

Without S9-mix

With S9-mix

                 Dose          Bacterial              Revertant

                (μg/plate)    background lawn  colonies

Dose       Bacterial                Revertant

(µg/plate) background lawn  colonies

TA1535

5000          slight                 -1

3330          extreme        microcolonies

5000          extreme        microcolonies

TA1537

3330          extreme        microcolonies

5000          extreme        microcolonies

3330          moderate           -1

5000          extreme        microcolonies

TA98

5000          moderate          -1to

                 to extreme    microcolonies

5000          -2                       -1

TA100

1000          moderate           moderate

3330          extreme        microcolonies

5000          extreme        microcolonies

1000          moderate           -1

3330          extreme        microcolonies

5000          extreme        microcolonies

-1  No reduction in the number of revertant colonies

-2      No reduction of the bacterial background lawn

 

Experiment 1: Mutagenic response of Sodium polysulfide solution in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain

Without S9-mixDose (µg/plate)

TA1535

TA1537

TA98

TA100

WP2uvrA

positive control

785 ± 42       

284 ± 34       

946 ± 60

883 ±  5

1086 ± 53

solvent control         

9 ±  1         

5 ±  3        

21 ±  2       

124 ± 12        

13 ±  2

3

 

 

 

115 ± 16        

20 ±  4

10

 

 

                                                  

113 ±  7        

21 ±  4

33        

11 ±  3         

6 ±  3        

17 ±  3       

109 ±  4        

18 ±  3

100         

8 ±  1         

5 ±  2        

21 ±  5       

116 ±  4        

20 ±  3

333         

6 ±  2         

5 ±  1        

26 ±  1       

128 ± 13        

20 ±  3

1000

5 ±  5 s       

7 ±  3        

29 ±  7       

141 ±  7 s      

18 ±  2

3330         

8 ±  2 m      

MC e      

MC e     

154 ± 13 m      

29 ±  5

5000        

MC e      

MC e

MC e

135 ± 15 m      

46 ±  9

With S9-mix1

 

 

 

 

 

positive control      

229 ±  7       

383 ± 30      

1026 ± 47      

1404 ± 11       

360 ±  5

solvent control         

5 ±  2         

3 ±  1        

25 ±  2       

110 ±  3        

16 ±  2

3

 

 

 

114 ± 18        

15 ±  5

10

 

 

 

112 ± 13        

16 ±  4

33         

7 ±  1         

3 ±  1        

24 ±  4       

106 ± 12        

17 ±  3

100

6 ±  2

3 ±  2

24 ±  4

110 ± 12

18 ±  2

333

6 ±  2

4 ±  2

27 ±  4

105 ±  6

14 ±  3

1000

7 ±  1

7 ±  1 s

35 ±  4

148 ± 15 s

18 ±  3

3330

MC      e      

MC      e      

40 ±  4       

141 ±  9 m      

35 ±  8

5000        

MC      e      

MC      e      

39 ± 14       

120 ±  1 m      

53 ±  3

Solvent control: 0.1 mlMilli-Q water

1    The S9-mix contained 5% (v/v) S9 fraction

s     Bacterial background lawn slightly reduced

m    Bacterial background lawn moderately reduced

e     Bacterial background lawn extremely reduced

MC  Microcolonies

 

Experiment 2: Mutagenic response of Sodium polysulfide solution in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay

                                               Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain

Without S9-mixDose (µg/plate)

TA1535

TA1537

TA98

TA100

WP2uvrA

positive control       

807 ± 40       

490 ± 34      

1081 ± 84      

1063 ± 27      

1216 ± 19

solvent control        

10 ±  4        

3 ±  1       

20 ±  2       

108 ±  7        

17 ±  3

33         

9 ±  5         

3 ±  1        

25 ±  2       

110 ± 10        

18 ±  4

100        

11 ±  6         

5 ±  2        

23 ±  2       

117 ± 15        

17 ±  3

333         

8 ±  2         

5 ±  2        

23 ±  8       

127 ± 14        

14 ±  4

1000         

7 ±  1         

6 ±  1        

31 ±  3        

61 ± 23 m      

26 ±  2

3330        

11 ±  5        

MC      e      

30 ±  6        

MC      e      

31 ±  5

5000

9 ±  3 s      

MC      e      

(2)             

MC      e      

54 ±  4

With S9-mix1

 

 

 

 

 

positive control       

167 ± 14       

312 ± 65       

685 ± 16      

1280 ± 30       

277 ±  8

solvent control

7 ±  3         

4 ±  1        

27 ±  4        

61 ±  5        

16 ±  2

33         

7 ±  2         

3 ±  0        

27 ±  2        

64 ±  9        

14 ±  5

100

3 ±  0

3 ±  1

25 ±  6

61 ± 10

14 ±  3

333

5 ±  2

4 ±  2

30 ±  3

61 ±  5

19 ±  4

1000

11 ±  3

4 ±  1

50 ±  6

73 ±  2 m

23 ±  5

3330

MC      e

5 ±  3 m

58 ±  5

MC      e

41 ±  6

5000        

MC      e      

MC      e      

52 ±  6        

MC      e      

62 ±  7

Solvent control: 0.1 ml Milli-Q water

1       The S9-mix contained 10% (v/v) S9 fraction

2       Two plates with 26 and 13 colonies showed a moderately reducedbacterial background lawn and one plate showed an extreme thinning of the microcolony lawn and an increase in the size of the microcolonies

s     Bacterial background lawn slightly reduced

m    Bacterial background lawn moderately reduced

e     Bacterial background lawn extremely reduced

MC  Microcolonies

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the absence of S9-mix, Sodium polysulfide solution induced dose related increases in tester strain WP2uvrA in two independently repeated experiments. The increases observed were above the laboratory historical control data range and 3.5 and 3.2-fold the concurrent control.

In the presence of S9-mix, Sodium polysulfide solution induced dose related increases in the tester strains TA98 and WP2uvrA. The increases observed in tester strain WP2uvrA were in two independently repeated experiments, were above the laboratory historical control data range and 3.3 and 3.9-fold the concurrent control. Although the increase observed in tester strain

TA98 was above the laboratory historical control data range, the increase was not three times the concurrent control (2.1-fold) and in the second experiment only. Therefore, this increase is considered to be not biologically relevant and Sodium polysulfide solution is considered to be only mutagenic in the Escherichia coli strain WP2uvrA.

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

Based on the results of this study it is concluded that Sodium polysulfide solution is not mutagenic in the Salmonella typhimurium reverse mutation assay and that Sodium polysulfide solution is mutagenic in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of Sodium polysulfide solution in theSalmonella typhimurium reverse mutation assay and theEscherichia colireverse mutation assay (with independent repeat).

 

Sodium polysulfide solution was tested in theSalmonella typhimurium reverse mutation assay with four histidine-requiring strains ofSalmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in theEscherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch 707-1 of Sodium polysulfide solution was a reddish brown liquid. The test substance was dissolved in Milli-Q water.

 

In the dose range finding test, Sodium polysulfide solution was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Sodium polysulfide solution did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 1000 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

 

Based on the results of the dose range finding test, Sodium polysulfide solution was tested in the first mutation assay at a concentration range of 33 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains, except in tester strain TA98 in the presence of S9-mix.

 

In an independent repeat of the assay with additional parameters, Sodium polysulfide solution was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains, except in the tester strains TA98 in the presence of S9-mix and WP2uvrA in the absence and presence of S9-mix.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

In the absence of S9-mix, Sodium polysulfide solution induced dose related increases in tester strain WP2uvrA in two independently repeated experiments. The increases observed were abovethelaboratory historical control data range and 3.5 and 3.2-fold the concurrent control.

 

In the presence of S9-mix, Sodium polysulfide solution induced dose related increases in the tester strains TA98 and WP2uvrA. The increases observed in tester strain WP2uvrA were in two independently repeated experiments, were abovethelaboratory historical control data range and 3.3 and 3.9-fold the concurrent control. Although the increase observed in tester strain TA98 was abovethelaboratory historical control data range, the increase was not three times the concurrent control (2.1-fold) and in the second experiment only. Therefore, this increase is considered to be not biologically relevant and Sodium polysulfide solution is considered to be only mutagenic in theEscherichia colistrain WP2uvrA.

  

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

 

Based on the results of this study it is concluded that Sodium polysulfide solution is not mutagenic in theSalmonella typhimurium reverse mutation assay and that Sodium polysulfide solution is mutagenic in the Escherichia coli reverse mutation assay