Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Feb 2019 - 14 Mar 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Identification: Zirconium (IV) acetylacetonate (Zr-acac)
- Description: White to off-white powder
- Storage conditions: At room temperature

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item formed a homogeneous suspension in DMSO and DMSO is accepted and approved by authorities and international guidelines
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9; 2.5 µg/plate in DMSO for TA1537 (direct plate assay)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9; 15 µg/plate in DMSO for TA1537 (pre-incubation assay), 10 µg/plate in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9; 10 µg/plate (direct plate assay) and 2 µg/plate (pre-incubation assay) in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9; 1 µg/plate (TA98 and TA100 (direct plate assay)), 2.5 µg/plate (TA1535, TA1537), 5 µg/plate (TA100 pre-incubation assay), 15 µg/plate (WP2uvrA) in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test item on the plates was determined.

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
- The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
- No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strain TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
- The negative response should be reproducible in at least one follow up experiment

A test item is considered positive (mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three times the concurrent control.
- In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.

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:
cytotoxicity
Vehicle 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:
cytotoxicity
Vehicle 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:
cytotoxicity
Vehicle 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:
cytotoxicity
Vehicle 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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation direct plate assay: precipitation was observed at the end of the incubation period at test item concentrations of 1600 µg/plate and upwards in tester strains TA100 and WP2uvrA in the absence and presence of S9-mix and at concentrations of 5000 µg/plate in tester strains TA1535, TA1537 and TA98 in the absence of S9-mix only. No precipitate was observed in tester strains TA1535, TA1537 and TA98 in the presence of S9-mix.
- Precipitation pre-incubation assay: precipitation of the test item was observed at the end of the incubation period at concentrations of 1600 µg/plate and upwards, except in tester strain TA100 in the absence of S9-mix and in tester strains TA1535, TA98 and TA100 in the presence of S9-mix where precipitate was observed at concentrations of 512 µg/plate and upwards. Since the test item precipitated heavily on the plates at the test item concentration of 1600 µg/plate in tester strains TA98 and TA100 in the presence of S9-mix and at the test item concentration of 5000 μg/plate in all the other tester strains, the number of revertants of these dose levels could not be determined.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 5000 µg/plate and with S9: 5000 µg/plate and above
TA1537: without S9: 500 µg/plate and with S9: 5000 µg/plate and above
TA98: without S9: 5000 µg/plate and above and with S9: 1600 µg/plate and above
TA100: without S9: 1600 µg/plate and above and with S9: 512 µg/plate and above
WP2uvrA: without S9: 5000 µg/plate and with S9: 5000 µg/plate and above

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles, Zirconium (IV) acetylacetonate was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed according to OECD guideline 471 and in accordance with GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. All bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitated was observed at the test item concentration of 1600 µg/plate in tester strains TA98 and TA100 in the presence of S9-mix and at the test item concentration of 5000 μg/plate in all the other tester strains. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Zirconium (IV) acetylacetonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.