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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:
2016-2-3 to 2016-6-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
[1,3-bis(2,4,6-trimethylphenyl)-4,5-dimethylimidazol-2-ylidene](2-thienylmethylidene)(tricyclohexylphosphine)ruthenium(II)dichloride
Cas Number:
1190427-50-9
Molecular formula:
C46H65Cl2N2PRuS
IUPAC Name:
[1,3-bis(2,4,6-trimethylphenyl)-4,5-dimethylimidazol-2-ylidene](2-thienylmethylidene)(tricyclohexylphosphine)ruthenium(II)dichloride
Test material form:
solid: particulate/powder

Method

Target gene:
S. typhimurium histidine (his) reversion system and the E. coli tryptophan (trp) reversion system.
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:
S9 mix
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
According to guideline, top dose is the recommended maximum test concentrationfor soluble non-toxic test compounds.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test compound is insoluble in water, DMSO was compatible with the survival of the bacteria used and with S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-experiment and Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: at least 48 h

SELECTION AGENT (mutation assays): L-histidine for S. typhimurium; tryptophan for E. coli

NUMBER OF REPLICATIONS: 3 plates per test condition

DETERMINATION OF CYTOTOXICITY
- Method: clearing/diminution of background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control
Rationale for test conditions:
test conditions were according to the standard OECD Guideline
Evaluation criteria:
A test was considered acceptable if for each strain:
- the bacteria demonstrated their typical responses to ampicillin (TA 98, TA 100)
- mean values of spontaneous reversion frequency of the negative control plates (A. dest.), with and without S9 mix, fell within the historical control data range (2012 -2014)
- corresponding background growth on both negative control and test plates was observed.
- the positive controls showed a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain could be analyzed

Evaluation of Mutagenicity:
The Mutation Factor was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control
A test item was considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occured and/or
- a biologically relevant positive response for at least one of the dose groups occured
in at least one tester strain with or without metabolic activation.

A biologically relevant increase as described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions was at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions was at least three times higher
when compared to the reversion rate of the solvent control.

According to the OECD guideline, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups iexamined, was considered to be non-mutagenic.

Results and discussion

Test resultsopen allclose all
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
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item did not exert cytotoxicity and, without metabolic activation, precipitation of the test item was observed in all tester strains used in experiment I and II at concentrations of 316 μg/plate and higher. With metabolic activation, precipitation was observed at concentrations of 1000 μg/plate and higher.

Any other information on results incl. tables

Please see attached file for results.

Applicant's summary and conclusion

Conclusions:
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in both a plate incorportion and a pre-incubation experiment. The reference mutagens induced a distinct increase of revertant colonies demonstrating the validity of the experiments and the functionality of the S9-mix used. Under the experimental conditions used, the test substance did neither cause gene mutations by base pair changes nor by frameshift in the genome of the tester strains used and is, therefore, considered to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

A valid bacterial reverse mutation assay according to OECD Guideline 471 and under GLP did not identify any signs of mutagenicity .

for genotoxicity according to OECD TG 471 and under GLP