Tetraammonium decachloro-μ-oxodiruthenate(4-)

Genetic toxicity: in vitro

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:

28 July - 21 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetraammonium-decachloro-mu-oxodiruthenate

IUPAC Name:

Tetraammonium-decachloro-mu-oxodiruthenate

Constituent 2

Reference substance name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

EC Number:

286-924-7

EC Name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

Cas Number:

85392-65-0

Molecular formula:

Cl10ORu2.4H4N

IUPAC Name:

tetraammonium decachloro-μ-oxodiruthenate(4-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): tetraammonium-decachloro-mu-oxodiruthenate
- Substance type: brown powder
- Physical state: solid
- Analytical purity: 98%
- Impurities (identity and concentrations): metallic impurities 0.3%
- Composition of test material, percentage of components: ((NH4)4Ru2Cl10O x H2O 98.0%, NH4Cl 0.7%, H2O 1.0%)
- Isomers composition: no data
- Purity test date: 15-Jul-2015
- Lot/batch No.: 71495159
- Expiration date of the lot/batch: 11-Jun-2016
- Stability under test conditions: no data
- Storage condition of test material: store in a cool, dry place in tightly closed containers at 5-25 deg C, shelf life 12 months from date of delivery

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range-finder experiment: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
Experiment 1: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
Experiment 2: 20.48, 51.2, 128, 320, 800, 2000 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: well-known solvent/vehicle

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Strain TA98 -S9 5 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 -S9 2 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 -S9 50 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA102 -S9 0.2 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 +S9 10 µg/plate

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100, TA1535, TA1537 +S9 5 µg/plate

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA102 +S9 20 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: Range-finder experiment (with and without S9), Experiment 1 (with and without S9) and Experiment 2 (without S9): in agar (plate incorporation); Experiment 2 (with S9): preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 3 days

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Evaluation criteria:

Acceptance Criteria: The assay was considered to be valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of ≥1.5 fold (in strain TA102), ≥2 fold (in strains TA98 and TA100) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S9 preparation.
Evaluation Criteria: For valid data, the test article was considered to be mutagenic if:
3. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values
4. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.

Statistics:

None

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in Experiment 1 (plate incorporation protocol): at 500 µg/plate, without S9; at 1600 µg/plate, with S9) (in Experiment 2: at 2000 µg/plate, without S9 (plate incorporation); at 5000 µg/plate, with S9 (pre-incubation))

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in Experiment 1 (plate incorporation protocol): at 500 µg/plate, without S9; at 1600 µg/plate, with S9) (in Experiment 2: at 2000 µg/plate, without S9 (plate incorporation); at 5000 µg/plate, with S9 (pre-incubation))

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Range-finder experiment: stock solution (filter sterilised), 20 mg Tetraammonium-decachloro-mu-oxodiruthenate/ml, pH 3; Experiment 1: pH of stock solution not measured (in error); Experiment 2: stock solution (not filter sterilised), 20 mg Tetraammonium-decachloro-mu-oxodiruthenate/ml, pH 2.19
- Precipitation: at 5000 µg/plate, Experiment 2 only

RANGE-FINDING/SCREENING STUDIES: strains TA98, TA100 and TA102; evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 5000 µg/plate in all strains in the absence and presence of S9

COMPARISON WITH HISTORICAL CONTROL DATA: mean vehicle control counts were comparable with the laboratory’s historical ranges; mean positive control counts were generally within or higher than the laboratory's historical control ranges

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

In a guideline bacterial reverse mutation (Ames) test, to GLP, tetraammonium-decachloro-mu-oxodiruthenate did not induce mutations in five histidine-requiring strains of Salmonella typhimurium, when tested up to 5 mg/plate in the absence or presence of a rat liver metabolic activation system (S9).

Executive summary:

Tetraammonium-decachloro-mu-oxodiruthenate was tested in a bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP.

The test substance was assayed in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of S. typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9), in two separate experiments (each in triplicate): in experiment 1, a plate incorporation protocol was used; the experiment was repeated, using an additional pre-incubation step for the test with S9. The highest concentrations of test article analysed were up to 5000 μg/plate or up to the limit of cytotoxicity and were determined following a preliminary toxicity range-finder experiment. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and fit the acceptance criteria.

There was no evidence of mutagenicity in any strain with or without S9 in either experiment. There was some evidence of toxicity at 500-5000 µg/plate with the plate incorporation protocol and at 2000-5000 µg/plate with the pre-incubation protocol. Vehicle and positive controls performed as expected.

It is concluded that tetraammonium-decachloro-mu-oxodiruthenate did not induce mutations in five strains of S. typhimurium when tested at concentrations up to 5000 μg/plate or up to the limit of toxicity, in the absence and in the presence of S9.