Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-11 to 2016-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
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

Constituent 1
Chemical structure
Reference substance name:
Strontium zirconium trioxide
EC Number:
234-847-4
EC Name:
Strontium zirconium trioxide
Cas Number:
12036-39-4
Molecular formula:
SrZrO3
IUPAC Name:
strontium dioxido(oxo)zirconium
Test material form:
solid: particulate/powder
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SZ-380-STM-16
- Expiration date of the lot/batch: 04 March 2017
- Purity: > 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions of test material: room temperature (15-25 ºC, below 70% relative humidity), protected from light and humidity

OTHER SPECIFICS:
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personal health and safety.
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 ethanol. 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 (TA98 and TA100, plate incorporation);
Initial mutation test: 5, 15.81, 50, 158.1, 500, 1581, 5000 µg/plate (all strains, plate incorporation);
Confirmatory mutation test: 5, 15.81, 50, 158.1, 500, 1581, 5000 µg/plate (all strains, pre-incubation).

The test item was found soluble in ethanol 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: ethanol
- Justification for choice of solvent/vehicle: In order to obtain a suitable solvent, a solubility test was performed using Milli-Q water, dimethyl sulfoxide (DMSO), ethanol, acetone, N,N-dimethylformamide (DMF) and 1 % methylcellulose. Each formulation was stirred in order to limit observed sedimentation, which rate depends on the solvent used. The following observations were made on the formulations at a 100 mg/mL concentration:
- Milli-Q water: fast sedimentation,
- DMSO: inhomogeneity and fast sedimentation,
- Ethanol: fine homogeneity and slower sedimentation,
- Acetone: immediate sedimentation,
- DMF: fast sedimentation,
- 1 % methycellulose: inhomogeneity with slow sedimentation.
Due to the homogeneity and the speed of the sedimentation, ethanol was selected as vehicle for the study.
The obtained stock solution (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 ethanol is presented in the section "Any other information on material and methods including tables".
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylenediamine
Remarks:
without S9; 4 µg/plate (TA98)
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)
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)
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)
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 dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions was 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 was 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:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
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 resultsopen allclose all
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
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Preliminary range finding 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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: fast sedimentation in formulation at 100 mg/mL
- Precipitation:
Preliminary Range Finding Test: slight precipitate at 5000 µg/plate with and without S9 (TA98, TA100)
Initial Mutation Test: slight precipitate at 5000 µg/plate with and without S9 (TA100, TA1535, TA1537)
Confirmatory Mutation Test: slight precipitate at 1581 and 5000 µg/plate with and without S9 (all strains)

RANGE-FINDING/SCREENING STUDIES
The observed number of revertant colonies was in the normal range. Sporadically, minor differences compared to the solvent control numbers were observed. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
Slight precipitate was observed in both tester strains with and without metabolic activation at the concentration of 5000 μg/plate.
Plates had white discoloration at the concentrations of 5000, 2500 and 1000 μg/plate in both tested strains with and without metabolic activation. This made the evaluation of the background lawn development difficult at 5000 μg/plate concentration.
Inhibitory or toxic effects of the test item were not detected in the Preliminary 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.

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/vehicle) and positive control plates were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY
No signs of inhibitory, cytotoxic effects of the test item (such as reduced background lawn development and/or reduced number of revertant colonies) were observed in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any concentration with or without metabolic activation.

Any other information on results incl. tables

Historical data for ethanol

No historical data are available for ethanol due to its infrequent usage as vehicle in Ames test. However, the mean values of revertant colony numbers of ethanol control plates in this study were within the general historical control range (including all the vehicles commonly used in Ames test).

Initial Mutation Test/Confirmatory Mutation Test

Plates showed white discoloration at the concentrations of 5000 and 1581 μg/plate in all tested strains with and without metabolic activation in the Initial Mutation Test and Confirmatory Mutation Test. The degree of the discoloration made the evaluation of the background lawn development difficult at 5000 μg/plate concentration in both main tests.

Slight decreases of the revertant counts were observed sporadically compared to the solvent control in the Initial Mutation Test and Confirmatory Mutation Test . However, the mean number of revertant colonies was within the historical control range, thus they were considered as biological variability of the test system.

Slight increases in the numbers of revertant colonies were detected compared to the solvent control in the Initial Mutation Test in Escherichia coli WP2 uvrA bacterial strain. However, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and were within the historical control range. They were considered as reflecting the biological variability of the test.

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 bacterial strains used.
In conclusion, the test item strontium zirconium trioxide has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.