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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16-Oct-2012 to 12-Nov-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed test according to OECD guideline 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(Adopted July 1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
- Name of test material (as cited in study report): Zirconium acetate solution
- Substance type: clear colourless solution
- Physical state: liquid
- Composition of test material, percentage of components: aqueous solution containing 40.7% zirconium acetate anhydrous, purity of dry content > 99%
- Lot/batch No.: 12/201
- Expiration date of the lot/batch: 31-Jul-2013
- Storage condition of test material: room temperature

Method

Target gene:
TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.

Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
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- 5,6 -Benzoflavone - induced rat liver S9 mix
Test concentrations with justification for top dose:
Preliminary assay:
The test item zirconium acetate solution was assayed in the toxicity test at a maximum concentration of zirconium acetate of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. The dose levels tested correspond to 2040, 647, 204, 64.7 and 20.4 µg zirconium/plate.
Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included.

Main Assay I (plate incorporation method): 2500, 1250, 625 and 313 µg of zirconium acetate/plate. The dose levels tested correspond to 2040, 1020, 511, 255 and 128 µg/plate of zirconium.

Main Assay II: 5000, 2500, 1250, 625 and 313 µg of zirconium acetate/plate. An additional dose level of 156 µg/plate was used with TA100 tester strain both in the absence and presence of S9 metabolism. The dose levels tested correspond to 2040, 1020, 511, 255, 128 and 63.9 µg/plate of zirconium.

Main Assay III:
- TA1535, TA1537, TA98 without S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 µg of zirconium acetate/plate
- TA100 without S9 at 78.1, 39.1, 19.5, 9.77, 4.88, 2.44, 1.22 µg of zirconium acetate/plate
- WP2 uvrA without S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88 µg of zirconium acetate/plate
- TA98 with S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88 µg of zirconium acetate/plate
- TA1535, TA1537, WP2 uvrA with S9 at 313, 156, 78.1, 39.1, 19.5, 9.77 µg of zirconium acetate/plate
- TA100 with S9 at 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 µg of zirconium acetate/plate
Vehicle / solvent:
The solvents used in this study were sterile water for injection and Dimethylsulfoxide (DMSO). Test item solutions were prepared in sterile water for injection.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
At 1 µg/plate without S9 in TA1535 and TA100 strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
At 50 µg/plate without S9 in TA1537 strain
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
At 2 µg/plate without S9 in TA98 strain
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
At 500 µg/plate without S9 in WP2 uvrA strain
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
At 1 µg/plate with S9 in TA 1535, TA 1537, TA 98, TA 100 and 10 µg/plate in WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
At 2 µg/plate with S9 in TA98 and TA 100 (pre-incubation method)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
At 20 µg/plate with S9 in WP2 uvrA (preincubation method)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Main assay: the first assay was performed using a plate-incorporation method. The second and third assays were performed using a pre-incubation method.

DURATION
- Preincubation period: in the second and the third assays, the incubate was vortexed and placed at 37°C for 30 minutes.
- Exposure duration: 72 hours ar 37°C

NUMBER OF REPLICATIONS: three replicate plates at each point including negative and positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was assessed in a prelimminary assay and on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

OTHER:
- Solutions of the test item, as received, were prepared immediately before use in sterile water for injection. Solutions were prepared on a weight/volume basis with correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of zirconium acetate and zirconium. All test item solutions were used within 2 hours and 45 minutes from the initial formulation. No assay of test item stability, nor its concentration and homogeneity in solvent were undertaken.

- Pre-incubation method: Two mL of overlay agar was added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

- Plate incorporation method: The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar (2 mL) and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

- The prepared plates in the plate-incorporation and pre-incubated methods were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, the scoring was effected by counting the number of revertant colonies on each plate.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Regression analysis:
1) The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as:
y = a + bx
where
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given)

2) The regression line includes the untreated control data.

3) Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation coefficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at all dose levels with all tester strains both in the absence and presence of S9 metabolism; further details specified in the field additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
-Solubility: Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at the zirconium acetate concentration of 50 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.
- Precipitation: Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations in Main assay I. Dose related precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period in Main assay II at the three highest concentrations. Precipitation in Main assay III is not reported.

RANGE-FINDING/SCREENING STUDIES: Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration. Slight toxicity, as indicated by thinning of the background lawn and reduction in revertant numbers, was observed with TA100 tester strain at the highest dose level, in the absence and presence of S9 metabolism.

COMPARISON WITH HISTORICAL CONTROL DATA: Results show that mean plate counts for untreated and positive control plates fell within the laboratory acceptance criteria based on historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Main assay I: Slight toxicity, as indicated by thinning of the background lawn, was observed with TA100 tester strain at the highest dose level both in the absence and presence of S9 metabolism.
- Main assay II: Marked toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed with all tester strains at all dose levels both in the absence and presence of S9 metabolism
-Main assay III: Dose related toxicity, as indicated by thinning of the background lawn, was observed at higher dose levels with all tester strains both in the absence and presence of S9 metabolism.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Sterility

The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly

Other

- The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation not mutagenic
negative without metabolic activation not mutagenic

It is concluded that the test item zirconium acetate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.