Sodium sulfide (Na2(Sx))

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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:

Sodium sulfide (Na2(Sx))

EC Number:

215-686-9

EC Name:

Sodium sulfide (Na2(Sx))

Cas Number:

1344-08-7

Molecular formula:

Na2Sx (x=1.5 - 5)

IUPAC Name:

disodium sulfanediide

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 / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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

Controls

Controls 1

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

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9

Migrated to IUCLID6: 60 µg/plate in milli-Q water for TA1537

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Controls 6

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 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

Positive controls validity:

valid

Test results 2

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-mix^1
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-mix^1
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