Reaction Mass of zirconium difluoride oxide and fluorozirconic acid

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:

2018-03-02 to 2018-04-30

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of zirconium difluoride oxide and fluorozirconic acid

EC Number:

947-897-3

Molecular formula:

(ZrOF2 )x(F6Zr.2H)y

IUPAC Name:

Reaction Mass of zirconium difluoride oxide and fluorozirconic acid

Test material form:

other: aqueous solution

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: Reaction Mass of zirconium difluoride oxide and fluorozirconic acid
- Source and lot/batch No.of test material: 18-KTS-003
- Expiration date of the lot/batch: no data
- Weight % water: 73.8%
- Weight % Zr: 11.5%
- Weight % solids: 26.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

Method

Target gene:

Histidine locus (Salmonella strains) and tryptophan locus (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate with and without S9-mix;
Confirmatory Mutagenicity Assay: 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate with and without S9-mix;

The dose levels in the initial toxicity-mutation assay were based on the pH results of two dilutions, conducted to assess the pH of the test substance once diluted.
Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test article and compatibility with the target cells.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9: 1.0 μg/plate for TA98, TA1535 ; 2.0 μg/plate for TA100, TA1537; 15 μg/plate for WP2 uvrA

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9: 1.0 μg/plate for TA98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9: 1.0 μg/plate for TA100, TA1535

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9: 75 μg/plate for TA1537

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9: 1000 μg/plate for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
One half (0.5) milliliter of S9 or sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle or test substance dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 60±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2C. Plates that were not counted immediately following the incubation period were stored at 2 8C until colony counting could be conducted.

DURATION
- Preincubation period: 60±2 minutes
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium) or Tryptophan (E. coli)

NUMBER OF REPLICATIONS:
Initial toxicity-mutation assay: duplicate
Confirmatory mutagenicity assay: triplicate


DETERMINATION OF CYTOTOXICITY
- Method: A dose level is considered toxic if one or both of the following criteria are met:
(1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose dependent drop in the revertant count.
(2) At least a moderate reduction in the background lawn.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

No formal hypothesis was done.
For each replicate plating, the mean and standard deviation of the number of revertants per plate
were calculated and are reported.

Results and discussion

Test results

Test results 1

Species / strain:

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

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

Test results 2

Species / strain:

E. coli WP2 uvr A

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance in water after pH adjustment formed solutions from 0.0300 to 1.00 mg/mL and workable suspensions from 3.00 to 100 mg/mL.
- Precipitation: Precipitate was observed at 5000 µg per plate with all conditions

RANGE-FINDING/SCREENING STUDIES:
The initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test substance, in duplicate, in the presence and absence of Aroclor induced rat liver S9.
No toxicity was observed. Precipitate was observed at 5000 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: all positive controls exhibited at least 3-fold increases in the number of revertants
- Negative (solvent/vehicle) historical control data: the number of revertants was within the characteristic ranges for all vehicle controls
All criteria for a valid study were met as described in the protocol.

Any other information on results incl. tables

Per the protocol, dilutions were conducted prior to the initiation of the initial-toxicity mutation assay to assess the pH of the test substance once diluted. In the first dilution, the pH of all test substance concentrations ranging from 5.00 and 100 mg/mL were measured using pH paper. They were all observed to be < 3.0.

In the second dilution, the initial pH of the top dilution at 100 mg/mL was 1.7. The pH of the top dose was adjusted with the drop-wise addition of 10N NaOH. The pH of the dilutions was measured using pH paper. The final pH of the top dose and the subsequent dilutions (ranging from 10.0 to 100 mg/mL) was 5.0.

Applicant's summary and conclusion

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate Reaction Mass of zirconium difluoride oxide and hexafluorozirconic acid was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.