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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10/9/2014-22/10/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

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:
Zirconium dinitrate oxide
EC Number:
237-529-3
EC Name:
Zirconium dinitrate oxide
Cas Number:
13826-66-9
Molecular formula:
N2O7Zr
IUPAC Name:
zirconium dinitrate oxide
Test material form:
solid
Details on test material:
Name of test material (as cited in study report): zirconium dinitrate oxide

Method

Target gene:
Histidine locus (Salmonella typhimurium)
Triptophan locus (Escherichia coli WP2 uvrA)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Preliminary concentration range finding test: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate
Based on the results of the preliminary experiment, the examined test concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate with and without metabolic activation.
Vehicle / solvent:
The following chemicals were used for vehicle (solvent) control groups:

Distilled water:
Supplier: TEVA Hungary Ltd.
Batch No.: 9321113
Expiry date: 30 November 2016

Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich Co.
Batch No.: SZBE0220V
Expiry date: 06 January 2017
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
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-phenylene diamine (NPD)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preliminary concentration range finding test, two independently performed main tests: in agar (plate incorporation); confirmatory mutation test: preincubation method.

DURATION
- Procedure for growing cultures: The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 µL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37°C in a Gyratory Water Bath Shaker.
- Exposure duration: 48 ± 1 hours
- Selection time (if incubation with a selection agent): 48 ± 1 hours

SELECTION AGENT (mutation assays): histidine for the Salmonella typhimurium strains and tryptophan for the Escherichia coli strain

NUMBER OF REPLICATIONS: In the test, each sample (including the controls) was tested in triplicate.

NUMBER OF CELLS EVALUATED:
The viability of each testing culture was determined by plating 0.1 mL of the 1E05, 1E06, 1E07 and 1E08 dilutions prepared by sterile physiological saline on Nutrient Agar plates. For each culture, the number of viable cells of the cultures was determined by manual counting after approximately 24-h incubation at 37°C.

DETERMINATION OF CYTOTOXICITY
- Inhibition of the background lawn of auxotrophic cells.
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, in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA strains, the number of reversions was more than twice higher than the spontaneous reversion rate of the negative (vehicle/solvent) control plates; the number of reversions was more than three times higher than the reversion rate of the negative (vehicle/solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
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 response in 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 software.

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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:
S. typhimurium TA 98
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:
S. typhimurium TA 100
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
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
- Solubility: The solubility of the test item was examined in a short solubility test using distilled water, dimethyl sulfoxide and N,N-dimethylformamide as vehicles. The test item was insoluble at 100 mg/mL in any of these vehicles (partial dissolution seen). However, the formulation at 50 mg/mL concentration using distilled water resulted in a homogeneous suspension, suitable for the test (while still partial dissolution was seen at the same concentration using the other two vehicles). Therefore, distilled water was selected as vehicle for the study.
- Precipitation: Precipitate/slight precipitate was detected on the plates in the main tests (initial mutation test and confirmatory mutation test) in all examined bacterial strains at 5000 and 1581 µg/plate concentrations with and without metabolic activation, and in the confirmatory mutation test at 500 µg/plate concentration with metabolic activation. However, the precipitate did not interfere with the scoring in those cases.

RANGE-FINDING/SCREENING STUDIES
In the preliminary concentration range finding test (informatory toxicity test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (± S9) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in this test. The number of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No signs of inhibitory, cytotoxic effects were observed in the preliminary experiment in any examined bacterial strain at any concentration with or without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range in all strains.

Any other information on results incl. tables

Validity of the tests:

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. Three replicates were used for each control and test item concentration. The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range in all strains. The examined concentration range was considered to be adequate as the test item was examined up to the recommended maximum concentration. At least five analyzable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Applicant's summary and conclusion

Conclusions:
In conclusion, the test item zirconium dinitrate oxide had no mutagenic activity in the examined bacterial strains under the test conditions of this study with and without metabolic activation.