Tungsten disulphide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-01-09, 2002-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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:

Tungsten

EC Number:

231-143-9

EC Name:

Tungsten

Cas Number:

7440-33-7

Molecular formula:

W

IUPAC Name:

tungsten

Constituent 2

Reference substance name:

Sulfur

EC Number:

231-722-6

EC Name:

Sulfur

Cas Number:

7704-34-9

Molecular formula:

S

IUPAC Name:

sulfur

Test material form:

solid: particulate/powder

Details on test material:

- Particle size distribution: D50= 3.1 um; D90=6.5 um
- Mass median aerodynamic diameter (MMAD):
- Density: 7.69 g/cm3

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16031/01
- Expiration date of the lot/batch: 01 April 2002
- Purity test date: >98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Dimethyl sulfoxide: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: suspended in dimethyl sulfoxide of spectroscopic quality (Merck). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension

FORM AS APPLIED IN THE TEST (if different from that of starting material)
stock solution

OTHER SPECIFICS:

Method

Target gene:

histidine-requiring Salmonella typhimurium
of tryptophan-requiring Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle:

Controls

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

methylmethanesulfonate

other: daunomycine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml}.
- Exposure duration: 48h
- Expression time (cells in growth medium): 20 min in medium (PIT) and incubation at 37C for 48-72 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix


NUMBER OF CELLS EVALUATED: 109cells/ml

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

?

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

?

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

?

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field "Test System"

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that tungsten disulphide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

Tungsten disulphide was tested in theSalmonella typhimuriumreverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in theEscherichia colireverse mutation assay with a tryptophan-requiring strain of Escherichia coliWP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). In the dose range finding test, tungsten disulphide was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Tungsten disulphide precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first and second mutation assay, tungsten disulphide was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. tungstn disulphide precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

Tungstn disulphide did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.  Based on the results of this study it is concluded that tungsten disulphide  is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay