oxozirconium; silane

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:

2017-08-23 to 2017-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

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:

oxozirconium; silane

EC Number:

811-319-7

Cas Number:

174633-44-4

Molecular formula:

(SiO2)x (ZrO2)y with 3% <= x <= 15% (w/w) 85% <= y <= 97% (w/w) H4OSiZr is the molecular formula for the hydrous form of the substance

IUPAC Name:

oxozirconium; silane

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Silicon zirconium oxide
- Physical state: solid
- Appearance: white powder
- Further information on test material confidential.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Based on the solubility test, a 100 mg/mL stock solution was prepared in DMSO. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10.

Method

Target gene:

Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537); tryptophan locus (E. coli strain WP2uvrA)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital-/β-naphthoflavone-induced rat liver post-mitochondrial S9 fraction

Test concentrations with justification for top dose:

Preliminary concentration range finding test: 10, 31.6, 100, 316, 1000, 2500, 5000 μg/plate with and without S9-mix (TA98 and TA100, plate incorporation);
Initial mutation test: 15.81, 50, 158.1, 500, 1581, 5000 μg/plate with and without S9-mix (all strains, plate incorporation);
Confirmatory mutation test: 5, 15.81, 50, 158.1, 500, 1581, 5000 μg/plate with and without S9-mix (all strains, pre-incubation).

The test item was found soluble in DMSO at 100 mg/mL (= 5000 μg/plate). Therefore, 5000 μg/plate was selected as top dose for the preliminary concentration range finding test. Based on the results of the range finding test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO), acetone and N,N-dimethylformamide (DMF). At 100 mg/mL concentration partial dissolution was observed in these vehicles. After 2 minutes ultrasonic water bath there was no change using distilled water, DMF and acetone but the test item was soluble using DMSO. DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension. The solubility of the test item in DMSO is presented in the section "Any other information on materials and methods including tables".

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylenediamine

Remarks:

without S9; 4 μg/plate (TA98)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; 2 μg/plate (TA100, TA1535)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9; 50 μg/plate (TA1537)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 2 μL/plate (WP2uvrA)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9; 2 μg/plate (TA98, TA100, TA1535, TA1537); 50 μg/plate (WP2uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Preliminary concentration range finding test/Initial mutation test: in agar (plate incorporation).
Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C). The content of the tubes was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

- Confirmatory mutation test: pre-incubation.
For the pre-incubation method, bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C. Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

DURATION
- Pre-incubation period: 48 h (confirmatory mutation test)
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 48 h (simultaneously with exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains); tryptophan (E. coli strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background inhibition; decrease in the number of revertant colonies

Evaluation criteria:

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft Excel TM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Remarks:

Preliminary range finding test, initial mutation test, and confirmatory mutation test.

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Preliminary range finding test and confirmatory mutation test. bacteria

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Partial dissolution observed at 100 mg/mL, even after 2 minutes ultrasonic water bath.
- Precipitation:
Preliminary Range Finding Test: No precipitate detected.
Initial Mutation Test: Precipitate/slight precipitate observed at 5000 μg/plate with and without S9-mix (all strains), and in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA strain with and without S9-mix at even the 1581 μg/plate concentration.
Confirmatory Mutation Test: Precipitate/slight precipitate observed at 5000 μg/plate with S9-mix (all strains), for Escherichia coli WP2 uvrA strain at even the 1581 μg/plate concentration and in all examined bacterial strains without S9-mix on the plates at 5000 and 1581 μg/plate concentrations.

RANGE FINDING/SCREENING STUDIES
The observed number of revertant colonies was in the normal range. Slight decreases of the revertant counts were observed compared to the solvent control sporadically. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
No precipitate of the test item was detected in the Preliminary Range Finding Test.
Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%))
- Positive historical control data: The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
- Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Inhibitory, cytotoxic effects of the test item were not detected in the Initial Mutation Test and Confirmatory Mutation Test.

Any other information on results incl. tables

Initial Mutation Test/Confirmatory Mutation Test

In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 158.1 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.31). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5000 and 15.81 μg/plate concentrations with metabolic activation (the observed mutation factor value was: MF: 1.60). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

 

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Additional information on precipitation

In the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1537 and Escherichia coli WP2uvrA strains with and without metabolic activation at 5000 µg/plate concentration the background lawn development could not be assessed due to the strong precipitate. In the Confirmatory Mutation Test in all Salmonella typhimurium strains with and without metabolic activation at 5000 µg/plate concentration and without metabolic activation at 1581 µg/plate concentration and in the Escherichia coli WP2uvrA strain with and without metabolic activation at 5000 and 1581 µg/plate concentration background lawn development could not be assessed due to the strong precipitate.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.