Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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

Constituent 1
Chemical structure
Reference substance name:
Tungsten disilicide
EC Number:
234-909-0
EC Name:
Tungsten disilicide
Cas Number:
12039-88-2
Molecular formula:
Si2W
IUPAC Name:
tungsten disilicide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch: 171130

Method

Target gene:
histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
Source of cells:
Tester strains TA98, TA1535 and TA102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA100 and TA1537 were obtained from Xenometrix AG, Switzerland.

MEDIA USED
Nutrient medium (per litre):
- 8 g Nutrient Borth
- 5 g NaCl
(solution of 125 μL ampicillin (10 mg/mL) (TA98, TA100, TA102) was added to retain phenotypic characteristics of strain)

Vogel-Bonner-salts (per litre)
- 10 g MgSO4 x 7 H2O
- 100 g citric acid
- 175 g NaNH4HPO4 x 4 H2O
- 500 g K2HPO4

Vogel-Bonner Medium E agar plates (per litre)
- 15 g Agar Agar
- 20 mL Vogel-Bonner salta
- 50 mL glucose-solution (40%)

Overlay agar (per litre)
- 7 g Agar Agar
- 6 g NaCl
- 10.5 mg L-histidine x HCl x H2O
- 12.2 mg biotin
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment.
5000 µg/plate was selected as the maximum concentration.
Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
A. dest
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Aqua dest.
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
- plate incorporation: Experiment 1; pre-incurbation: Experiment 2

EXPERIMENTAL PERFORMANCE
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

The concentrations, including the controls, were tested in triplicate.

EVALUATION OF CITOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
-a clear and dose-related increase in the number of revertants occurs and/or
-a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
-if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
-if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control [11].
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is notregarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA102 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I (with and without metabolic activation). In experiment II precipitation was observed in all tester strains used (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Tungsten Disilicide at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Any other information on results incl. tables

Pre-experiment for toxicity and Main experiments I and II: Result tables are attched in box "Attached background material"

Applicant's summary and conclusion

Conclusions:
Tungsten Disilicide is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA102) were exposed to Tungsten disilicide (> 88% purity) in DMSO at concentrations of 31.6, 100, 316, 1000, 2000 and 5000 μg/plate (experiment 1: plate incorporation, experiment 2: pre-incubation) in the presence and absence of mammalian metabolic activation.

Tungsten disilicide was tested up to the limit dose (5.0 mg/plate). Tungsten disilicide did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.