Tetraammonium decachloro-μ-oxodiruthenate(4-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation (Ames) test, conducted according to OECD Test Guideline 471 and to GLP, tetraammonium-decachloro-mu-oxodiruthenate failed to induce an increase in mutation frequency in five histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537 and TA102) when tested at concentrations of up to 5000 μg/plate or up to the limit of cytotoxicity, in the absence and presence of a rat liver metabolic activation system (S9) (McGarry, 2016).

 

In an in vitro mammalian cell gene mutation assay, conducted in accordance with OECD Test Guideline 476 and to GLP, tetraammonium-decachloro-mu-oxodiruthenate did not induce biologically relevant increases in mutations at the hprt locus of mouse lymphoma L5178Y cells when tested up to precipitating concentrations in the absence and presence of S9 (Lloyd, 2016a).

 

In an in vitro micronucleus assay, conducted according to OECD Test Guideline 487 and to GLP, tetraammonium-decachloro-mu-oxodiruthenate failed to induce increases in micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of solubility, for 3 hours in the absence and presence of S9, and for 24 hours without S9 (Lloyd, 2016b).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

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

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

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

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

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

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

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

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

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

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'.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 November 2015 - 24 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Growth media (RPMI 1640) prepared from horse serum, penicillin, streptomycin, amphotericin B, sodium pyruvate acid and pluronic.
- Properly maintained: yes. The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells were stored as frozen stocks in liquid nitrogen.
- Periodically checked for Mycoplasma contamination: yes. Each batch of frozen cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: yes. Each batch of frozen cells was purged of mutants

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver post-mitochondrial fraction (S-9) from male Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Cytotoxicity range-finder experiment: 62.5, 125, 250, 500, 1000 and 2000 μg/mL (both with and without S9)
Experiment 1: 100, 200, 400, 600, 800, 1000 and 1200 μg/mL (without S9) and 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600 and 1800 μg/mL (with S9)
Experiment 2: 125, 250, 500, 750, 1000, 1200 and 1400 μg/mL (with and without S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Purified water diluted 10-fold in the treatment medium
- Justification for choice of solvent/vehicle: No data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

4-nitroquinoline 1-oxide (NQO; concentrations of 0.15 and 0.20 μg/mL without S9) and benzo[a]pyrene (B[a]P; concentrations of 2.00 and 3.00 μg/mL with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. At least 10^7 cells in a volume of 17 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle or test article or 0.2 mL positive control solution (plus 1.8 mL purified water) was added. In addition, S-9 mix or 150 mM KCl was added. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL. After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 x g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 x g) for 5 minutes and resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.

DURATION
- Preincubation period: No data
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: For survival (192 wells at 1.6 cells/well), for viability (192 wells at 1.6 cells/well) and for 6TG resistance (384 wells at 20000 cells/well).

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test substance was measured by calculating the relative survival percentages.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable

Evaluation criteria:

The assay was considered valid if both the mutant frequency (MF) in the vehicle control cultures fell within the normal range (up to three times the historical control value) and at least one concentration of each of the positive control chemicals induced a clear, unequivocal increase in MF.
For valid data, the test article was considered to induce forward mutations at the hprt locus if: a) the MF at one or more of the concentrations was significantly greater than that of the vehicle control (p<=0.05); b) there was a significant concentration-relationship as indicated by the linear trend analysis (p<=0.05); c) the effects were reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Dunnett's test (one-sided) was used for the analysis of the statitical significance of increased mutant frequencies at different concentrations relative to controls.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest concentrations analysed in experiment 1 (1200 (-S9) and 1800 (+S9) μg/mL) gave 100 and 69% RS respectively. The highest concentrations analysed in experiment 2 (1400 μg/mL, - and + S9) gave 25 and 27% RS respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations tested (2000 μg/mL) as compared to the concurrent vehicle controls.
- Evaporation from medium: No data
- Water solubility: Preliminary solubility data indicated that the test item was soluble in purified water at concentrations up to approximately 20.00 mg/mL. The solubility limit in culture medium was in excess of 2000 μg/mL.
- Precipitation: Observed at 2000 μg/mL in the range-finder experiment. Following the 3 hour treatment incubation period in experiment 1, precipitate was observed at the highest five concentrations tested in the absence of S-9 (1200 to 2000 μg/mL) and the highest two concentrations tested in the presence of S-9 (1800 to 2000 μg/mL). In experiment 2 (following 3-hr treatment), precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (1400 to 2000 μg/mL). In both experiments, the lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded.

RANGE-FINDING/SCREENING STUDIES:

MUTAGENICITY (INCLUDING COMPARISON WITH HISTORICAL CONTROL DATA): In the absence and presence of S-9 in Experiment 1 and in the presence of S-9 in Experiment 2, no significant increases in MF were observed at any of the test item concentrations analysed. A statistically significant linear trend (p≤0.001) was seen in the absence of S-9 in Experiment 1, but in the absence of any statistically significant increases in MF at any concentration analysed, this observation was considered not biologically relevant.

In the absence of S-9 in Experiment 2, a statistically significant increase in MF over the concurrent control was observed at the highest concentration analysed (1400 μg/mL), and there was a weakly significant linear trend (p≤0.05). The mean MF value at 1400 μg/mL was 4.34 mutants per 10^6 viable cells, compared to 0.87 for the vehicle control. The historical mean MF for the vehicle control was 3.21 and therefore vehicle control MF values of up to 9.63 (3.21 x 3) would have been acceptable in this experiment. The MF value at 1400 μg/mL was only 1.35-fold greater than 3.21 and the concurrent vehicle control MF was low, therefore the increase observed at 1400 μg/mL was significant despite being small in magnitude. Furthermore, there was no evidence of reproducibility between experiments in the absence of S-9. Although statistically significant linear trends were observed in the absence of S-9 in both experiments, the small increase in MF observed in Experiment 2 was considered not biologically relevant.

MF values in vehicle control cultures fell within acceptable ranges and increases in mutation were induced by the positive control chemicals NQO (without S-9) and B[a]P (with S-9). However, the positive control responses for B[a]P in the presence of S-9 in Experiment 2 were lower than expected and the response observed at 2 μg/mL was not acceptable (less than a 2-fold increase over the vehicle control). The analogous response at 3 μg/mL was over 2-fold greater than the concurrent mean vehicle control MF and more than three times the corresponding historical mean vehicle control value. Therefore the study was accepted as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The highest concentrations analysed in experiment 1 (1200 and 1800 μg/mL without and with S9 respectively) gave 100 and 69% RS respectively. The highest concentrations analysed in experiment 2 (1400 μg/mL, - and + S9) gave 25 and 27% RS respectively. See tables 1 and 2 for %RS values for the individual concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In a guideline in vitro mammalian cell gene mutation assay, to GLP, tetraammonium-decachloro-mu-oxodiruthenate did not induce biologically relevant increases in mutations at the hprt locus of mouse lymphoma L5178Y cells when tested up to precipitating concentrations in the absence and presence of S9.

Executive summary:

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 476 (in vitro mammalian cell gene mutation assay), tetraammonium-decachloro-mu-oxodiruthenate was tested for its ability to induce gene mutations at the hprt locus in mouse lymphoma L5178Y cells.

In a cytotoxicity range finding study, six concentrations (62.5-2000 μg/mL) were tested (with and without S9); precipitation was seen after 3 hours incubation at the highest concentration. In the main test, cells were exposed to test material for 3 hr in two independent experiments, each in the absence and presence of S9. The highest concentrations analysed, limited by precipitation, were 1200 μg/mL and 1800 μg/mL (Experiment 1, without and with S9, respectively) exhibiting 100% and 69% relative survival (RS), respectively. In Experiment 2, the highest concentrations analysed was 1400 μg/mL, both without and with S9), which gave RS values of 25% and 27%, respectively.

No significant increases in mutant frequency (MF) over the concurrent controls were observed following treatment with tetraammonium-decachloro-mu-oxodiruthenate at any concentration analysed in the absence and presence of S9 in Experiment 1 and in the presence of S9 in Experiment 2. Although a statistically significant increase in MF was observed at the highest concentration analysed in the absence of S9 in Experiment 2 (1400μg/mL), this was considered not biologically relevant since the concurrent vehicle control MF was low compared with the historical control and there was a lack of reproducibility between experiments. The statistically significant linear trends observed in both experiments were also considered not biologically relevant.

Overall, tetraammonium-decachloro-mu-oxodiruthenate did not induce biologically relevant increases in gene mutations at the hprt locus of L5178Y mouse lymphoma cells, when tested up to the limits of solubility in two independent experiments, each in the presence and absence of S9.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July - 28 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (2014). In Vitro Mammalian Cell Micronucleus Test

Deviations:

yes

Remarks:

(no historical positive control data presented; this is not considered to detract from the quality of the study)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human, whole blood

Details on mammalian cell type (if applicable):

- Type and identity of media: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin/ streptomycin

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: 7.256-2000 µg/mL
Micronucleus experiment (3+21): 25-750 µg/mL
Micronucleus experiment (24+0): 50-1000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

3+21 hours, -S9, 0.3 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

3+21 hours, +S9, 3.0 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

aneugen

Positive control substance:

other: noscapine

Remarks:

24+0 hours, -S9, 30 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: range-finder experiment: 3 or 24 hours; micronucleus experiment: 3 or 24 hours
- Expression time (cells in growth medium): range-finder experiment: 21 or 24 hours (24 hours in error, 0 hours intended, protocol deviation), respectively; micronucleus experiment: 21 or 0 hours, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours for all cultures

SPINDLE INHIBITOR (cytogenetic assays): for 3+21 cultures, 20 hours (cytochalasin B added to post-treatment medium (post-treatment wash procedure takes approx 1 hour)); for 24+0 cultures: 24 hours (cytochalasin B added at the time of treatment)
STAIN (for cytogenetic assays): acridine orange

NUMBER OF CELLS EVALUATED: 1000/culture (2000/concentration); except vehicle control for 3+21 hour cultures +S9, 4000 (1000/culture)

DETERMINATION OF CYTOTOXICITY
- Method: other: replication index, RI, the relative number of nuclei compared to vehicle controls (((number of binucleate cells + 2(number of multinucleate cells))/(total number of cells in treated cultures)); relative RI as % for each treated culture (((RI of treated cultures)/(RI of vehicle control cultures))*100)

OTHER EXAMINATIONS:
- Other: Slide Analysis
Scoring was carried out using fluorescence microscopy.
Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

Evaluation criteria:

Acceptance Criteria
The assay was considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of micronucleated binucleate (MNBN) cell frequency) between replicate cultures, particularly where no positive responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the 95th percentile of the current observed historical vehicle control (normal) ranges
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range.
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at
the time of harvest
5. The maximum concentration analysed under each treatment condition was the lowest precipitating concentration

Evaluation Criteria
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Statistics:

The proportions of micronucleated binucleate (MNBN) cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson C, Williams D A, Allen J A, Amphlett G, Chanter D O and Phillips B (1989). Analysis of data from in vitro cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Sub-committee Report, Part III), Ed D J Kirkland, Cambridge University Press, pp 141-154). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

Species / strain:

lymphocytes: human, whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

3+21 hours -/+S9; 24+0 hours -S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH were observed at the highest concentration tested in the Range-Finder Experiment (2000 μg/mL), compared to the concurrent vehicle controls. Based on the concentrations tested in the Micronucleus Experiment, no further measurements of pH were deemed necessary.
- Effects of osmolality: No marked changes in osmolality were observed at the highest concentration tested in the Range-Finder Experiment (2000 μg/mL), compared to the concurrent vehicle controls. Based on the concentrations tested in the Micronucleus Experiment, no further measurements of osmolality were deemed necessary.
- Water solubility: soluble at concentrations up to 20 mg/mL in water; the solubility limit in culture medium was in excess of 2000 μg/mL.
- Precipitation: a lack of visible precipitation at 2000 µg/ml immediately upon test article addition or following an incubation period of approximately 20 hours. In the micronucleus experiment, the highest concentration selected for scoring was the lowest precipitating concentration at the end of the incubation period, 400 µg/mL .

RANGE-FINDING/SCREENING STUDIES: cytotoxicity:
3+21 hours, -S9: up to 93 µg/mL, 0%; 156 µg/mL, 7%; 259 µg/mL, 6%, 432 µg/mL, 9%; 720 µg/mL, 21%; 1200 µg/mL and above, not evaluated.
3+21 hours, +S9: up to 56 µg/mL, 0%; 93 µg/mL, 6%; 156 µg/mL, 7%; 259 µg/mL, 6%, 432 µg/mL, 6%; 720 µg/mL, 4%; 1200 µg/mL, 30%; 2000 µg/mL, not evaluated.
24+24 hours, +S9: up to 34 µg/mL, 0%; 56 µg/mL, 3%; 93 µg/mL, 9%; 156 µg/mL, 7%; 259 µg/mL, 14%, 432 µg/mL, 19%; 720 µg/mL, 26%; 1200 µg/mL, 49%; 2000 µg/mL, not evaluated.

COMPARISON WITH HISTORICAL CONTROL DATA: frequency of micronucleated binucleate (MNBN) cells/cells scored(%):
Vehicle control
3+21 hours, -S9: mean, 0.64, 95% reference range, 0.20-1.40
3+21 hours, +S9: mean, 0.62, 95% reference range, 0.20-1.20
24+0 hours, -S9: mean, 0.68, 95% reference range, 0.20-1.20
Positive control
no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: micronucleus experiment:
3+21 hours, -S9: up to 175 µg/mL, not evaluated; 200, 250 µg/mL, 0%; 300 µg/mL, 2%, 400 µg/mL, 7%; 500 µg/mL, 9%; 750 µg/mL, 16%.
3+21 hours, +S9: up to 200 µg/mL, not evaluated; 250-750 µg/mL, 0%.
24+0 hours, -S9: 50, 100 µg/mL, 3%; 150 µg/mL, 2%; 200 µg/mL, 7%; 250 µg/mL, 6%; 300 µg/mL, 8%, 350 µg/mL, 2%; 400 µg/mL, 3%; 450 µg/mL, 2%; 500 µg/mL, 7%; 750 µg/mL, 16%; 1000 µg/mL, 20%.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In a guideline in vitro micronucleus assay, to GLP, tetraammonium-decachloro-mu-oxodiruthenate failed to induce increases in micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of solubility in the absence and presence of S9.

Executive summary:

Tetraammonium-decachloro-mu-oxodiruthenate was tested for its ability to induce chromosome damage (micronuclei) in human peripheral blood lymphocytes in a guideline study, to GLP.

The highest concentrations of test article analysed were limited by precipitation and were determined following a preliminary range-finder experiment. Cells were treated with the test material (at up to 400 µg/plate, the limit of solubility) for either 3 hours (with 21 hours recovery time) in the presence and absence of S9, or for 24 hours without S9. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and matched the acceptance criteria.

There was no evidence of an increase in micronucleus frequency with or without S9 following treatment of cells with tetraammonium-decachloro-mu-oxodiruthenate for 3 hours and/or 24 hours. Some evidence of cytotoxicity was observed at 100 µg/mL without S9, but none with S9. Vehicle and positive controls performed as expected.

Overall, tetraammonium-decachloro-mu-oxodiruthenate did not induce increases in the frequency of micronuclei in human peripheral blood lymphocytes treated in culture, when tested up to the limit of solubility in the presence and absence (3-hour treatment) or absence (24-hour treatment) of S9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity data were identified.

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

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 mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) 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 (McGarry, 2016).

 

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 476 (in vitro mammalian cell gene mutation assay), tetraammonium-decachloro-mu-oxodiruthenate was tested for its ability to induce gene mutations at the hprt locus in mouse lymphoma L5178Y cells. In a cytotoxicity range finding study, six concentrations (62.5-2000 μg/mL) were tested (with and without S9); precipitation was seen after 3 hours incubation at the highest concentration. In the main test, cells were exposed to test material for 3 hr in two independent experiments, each in the absence and presence of a rat liver metabolic activation system (S9). The highest concentrations analysed, limited by precipitation, were 1200 μg/mL and 1800 μg/mL (Experiment 1, without and with S9, respectively) exhibiting 100% and 69% relative survival (RS), respectively. In Experiment 2, the highest concentrations analysed was 1400 μg/mL, both without and with S9), which gave RS values of 25% and 27%, respectively. No significant increases in mutant frequency (MF) over the concurrent controls were observed following treatment with tetraammonium-decachloro-mu-oxodiruthenate at any concentration analysed in the absence and presence of S9 in Experiment 1 and in the presence of S9 in Experiment 2. Although a statistically significant increase in MF was observed at the highest concentration analysed in the absence of S9 in Experiment 2 (1400μg/mL), this was considered not biologically relevant since the concurrent vehicle control MF was low compared with the historical control and there was a lack of reproducibility between experiments. The statistically significant linear trends observed in both experiments were also considered not biologically relevant. Overall, tetraammonium-decachloro-mu-oxodiruthenate did not induce biologically relevant increases in gene mutations at the hprt locus of L5178Y mouse lymphoma cells, when tested up to the limits of solubility in two independent experiments, each in the presence and absence of S9 (Lloyd, 2016a).

 

Tetraammonium-decachloro-mu-oxodiruthenate was tested for its ability to induce chromosome damage (micronuclei) in human peripheral blood lymphocytes in an assay conducted in accordance with OECD Test Guideline 487 and to GLP. The highest concentrations of test article analysed were limited by precipitation and were determined following a preliminary range-finder experiment. Cells were treated with the test material (at up to 400 µg/plate, the limit of solubility) for either 3 hours (with 21 hours recovery time) in the presence and absence of S9, or for 24 hours without S9. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and matched the acceptance criteria. There was no evidence of an increase in micronucleus frequency with or without S9 following treatment of cells with tetraammonium-decachloro-mu-oxodiruthenate for 3 hours and/or 24 hours. Some evidence of cytotoxicity was observed at 100 µg/mL without S9, but none with S9. Vehicle and positive controls performed as expected. Overall, tetraammonium-decachloro-mu-oxodiruthenate did not induce increases in the frequency of micronuclei in human peripheral blood lymphocytes treated in culture, when tested up to the limit of solubility in the presence and absence (3-hour treatment) or absence (24-hour treatment) of S9 (Lloyd, 2016b).

Justification for classification or non-classification

Based on the existing in vitro genotoxicity data set, tetraammonium-decachloro-mu-oxodiruthenate does not meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008).