Tungsten Oxide (WO3), caesium and tin-doped

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:

27 June 2017 to 31 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tungsten Oxide (WO3), caesium and tin-doped

EC Number:

945-942-1

Molecular formula:

Cs0.29Sn0.04WO3

IUPAC Name:

Tungsten Oxide (WO3), caesium and tin-doped

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Dark blue powder

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain 1

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: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-range Finding Test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
First Experiment: Direct Plate Assay: 52, 164, 512, 1600 and 5000 μg/plate
Second Experiment: Pre-Incubation Assay: 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material could not be dissolved or homogenously suspended in Milli-Q water, DMSO, Ethanol, Acetone or Hexane. Based on the results of the solubility experiment, DMSO was selected as the most appropriate vehicle. At 50, 25 and 10 mg/mL a non-homogenous saturated suspension was obtained, in which a small amount of the test material remained on the bottom of the tube. All test material formulations were prepared separately and formed a non-homogenous saturated suspension, except for the concentrations of 0.054 and 0.017 mg/mL, where the appearance of the stock solution could not be determined due to the low test material concentrations. All stock solutions were treated with ultrasonic waves. Due to the solubility of the test material, the actual test concentrations are not known since the test material could not be homogeneously suspended or dissolved completely in DMSO. However, since the undiluted fraction of the test material is considered to be low, the deviation from the proposed concentration is expected to be low. Therefore, the concentrations are reported as proposed.
- Test material concentrations were used within 4 hours after preparation.
- Any residual volumes were discarded.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 and 2-aminoanthracene

Details on test system and experimental conditions:

DOSE RANGE FINDING TEST
- Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
-The highest concentration of the test material used in the subsequent mutation assays was 5000 μg/plate.

FIRST EXPERIMENT: DIRECT PLATE ASSAY
- The dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test material was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
- The test material was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37 °C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

INCUBATION AND SCORING
- After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted. The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data at the laboratory.
- The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
- No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

INTERPRETATION
A test material is considered negative (not mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
- The negative response should be reproducible in at least one follow up experiment.
A test material is considered positive (mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
- In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

FIRST EXPERIMENT: DIRECT PLATE ASSAY
- Precipitate: Precipitation of the test material on the plates was not observed at the start of the incubation period. At the end of the incubation period, the test material precipitated at the concentration of 5000 µg/plate.
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed. In tester strain TA1537, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 5000 µg/plate in the absence of S9-mix. However, since the reduction in the mean number of revertant colonies was only minor when compared against relevant historical control data and no toxicity was observed in any of the other tester strains in both experiments, this reduction is caused by an incidental fluctuation in the number of revertant colonies and is considered as not biologically relevant.
- Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested. In tester strain TA100, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the absence and presence of S9-mix. Since no dose-relationship was observed and the increases were not two-fold (a maximum of 1.2-fold reached), these increases were not considered to be biologically relevant.

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
- Precipitate: Precipitation of the test material on the plates was not observed at the start of the incubation period. At the end of the incubation period, the test material precipitated at the concentration of 5000 µg/plate.
- Toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
- All 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.
- 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.

Any other information on results incl. tables

Table 1: Summary of Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 52 164 512 1600 5000	148 165 162 138 150 142	9 10 12 9 15 10	27 24 19 38 32 21	14 14 13 21 10 12	7 4 4 4 3 2
+	Solvent 52 164 512 1600 5000	149 161 149 156 137 142	11 14 12 13 11 12	28 33 30 21 23 29	22 20 24 19 16 20	9 8 4 7 12 6
Positive Controls
-	Name	MMS	SA	4NQO	2NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	1025	887	1079	1272	1008
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2.5	15	1	2.5
	Mean no. colonies/plate	1268	263	402	1055	366

2AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

MMS = methylmethanesulfonate

4NQO= 4-nitroquinoline N-oxide

Table 2: Summary of Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 52 164 512 1600 5000	93 100 98 100 93 99	11 9 10 7 7 8	31 30 29 29 34 36	15 17 13 12 14 12	6 4 6 8 7 5
+	Solvent 52 164 512 1600 5000	90 92 93 96 101 99	10 8 14 9 9 9	34 40 42 38 40 42	21 21 18 22 19 19	8 9 8 8 5 4
Positive Controls
-	Name	MMS	SA	4NQO	2NF	2NF
	Concentration (µg/plate)	650	5	10	10	15
	Mean no. colonies/plate	822	879	153	1314	167
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	5	2.5	15	1	2.5
	Mean no. colonies/plate	1482	190	555	431	130

2AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

MMS = methylmethanesulfonate

4NQO= 4-nitroquinoline N-oxide

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay in the presence or absence of metabolic activation.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The objective of this study was to determine the potential of the test material to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The test was performed in two independent experiments, a direct plate assay and a pre-incubation assay.

The vehicle of the test material was dimethyl sulfoxide. Actual test concentrations are not known since the test material could not be homogeneously suspended or dissolved completely in dimethyl sulfoxide. However, since the undiluted fraction of the test material was considered to be low, the deviation from the proposed concentration is expected to be low.    

In the dose-range finding study, the test material was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material precipitated on the plates at the dose level of 5000 μg/plate. 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 mutation experiment, the test material was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test material precipitated on the plates at the dose level of 5000 μg/plate. 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 second mutation experiment, the test material was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test material precipitated on the plates at the dose level of 5000 μg/plate. 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. 

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.

The test material did not induce a significant dose-related 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. These results were confirmed in a follow-up experiment.

Under the conditions of the study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay in the presence or absence of metabolic activation.