Ruthenium trichloride hydrate

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, ruthenium trichloride hydrate 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 S9 (McGarry, 2016).

 

In an in vitro mammalian cell gene mutation assay, conducted in accordance with OECD Test Guideline 476 and to GLP, ruthenium trichloride hydrate failed to induce biologically relevant increases in mutations at the hprt locus of L5178Y mouse lymphoma cells when tested up to precipitating concentrations in two independent experiments, each 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, ruthenium trichloride hydrate 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).

An expert review of the genotoxicity data of Ruthenium Trichloride (hydrate) [CAS 14898-67-0; EC 604-667-4] is included in IUCLID Section 13.

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:

16 Sep - 9 Nov 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 and mutation experiment 1: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate.
Mutation experiment 2: 160, 300, 625, 1250, 2500 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:

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; TA102 +S9 20 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: Toxicity Range-Finder Experiment, Mutation Experiment 1: in agar (plate incorporation); Mutaion Experiment 2: preincubation

DURATION
- Preincubation period: Mutation Experiment 2: 20 minutes
- Exposure duration: 3 days

DETERMINATION OF CYTOTOXICITY
- Method: plates were examined for evidence of toxicity to the background lawn

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. 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
2. 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 [n]either 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 Mutation Experiment 1: from 500 to 5000 µg/plate -S9 and from 1600-5000 µg/plate +S9; Mutation Experiment 2: 2000 and/or 5000 µg/plate -S9 and +S9

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 Mutation Experiment 1: from 500 to 5000 µg/plate -S9 and from 1600-5000 µg/plate +S9; Mutation Experiment 2: 2000 µg/plate -S9 and +S9

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: pH of 50 mg/mL stock formulations was 0.79-0.93.
- Effects of osmolality: no data
- Water solubility: Preliminary solubility data indicated that Ruthenium (III) chloride hydrate was soluble in water for irrigation (purified water) at concentrations equivalent to at least 50 mg/mL.
- Precipitation: Toxicity Range-Finder Experiment and Mutation Experiment 1: no precipitation; Mutation Experiment 2: precipitation at 625 µg/plate and above in all strains in the presence of S9 only

RANGE-FINDING/SCREENING STUDIES: An initial toxicity Range-Finder Experiment was carried out in the absence and in presence of S9 in strains TA98, TA100 and TA102, using final concentrations of Ruthenium (III) chloride hydrate at 5, 16, 50, 160, 500, 1600, and 5000 μg/plate, plus vehicle and positive controls. Following these treatments evidence of toxicity was observed at 1600 and/or 5000 μg/plate in all strains in the absence and presence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the laboratory’s historical ranges; mean positive control counts fell within or slightly above the laboratory's historical control ranges, except for Mutation Experiment 2, TA1535 +S9 for which the mean positive control count was 115.7 and the historical control range was 127-398 (the effect was, nevertheless, clearly positive).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In a guideline study, to GLP, ruthenium (III) chloride hydrate 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:

Ruthenium trichloride hydrate 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 (S9), in two separate experiments (each in triplicate). 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. In experiment 1 a plate incorporation protocol was used; the experiment was repeated using a pre-incubation step. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition.

There was no evidence of mutagenicity in any strain with or without S9 in either experiment. Some evidence of toxicity was observed at 1600 and/or 5000 µg/plate with the plate incorporation protocol and at 1250 and/or 2500 µg/plate with the pre-incubation protocol. Vehicle and positive controls performed as expected. It is concluded that ruthenium trichloride hydrate 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.

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:

3 December 2015 - 6 March 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:

yes

Remarks:

In error, pH measurements were not taken for the stock formulation used in Experiment 2 of this study.

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, pyruvic acid and pluronic.
- Properly maintained: yes. The master stock of L5178 tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance 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: 12.5, 25, 50, 100, 200 and 300 μg/mL (without S9) and 12.5, 25, 50, 100, 200, 300, 400 and 500 μg/mL (with S9)
Experiment 2: 25, 50 100, 150, 200, 250, 300 and 400 μg/mL (with and without S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Solubility trials indicated that the test article was solule in water for irrigation at a concentration of 56.15 mg/mL

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 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): 12-13 days.
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG).

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: 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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

No precipitate was observed at the time of treatment but following the 3 hour incubation period, precipitate was observed at the highest three concentrations in the absence and presence of S9 (500 to 2000 μg/mL in the cytotoxicity range finder experiment)

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 and osmality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations tested (500 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 56.15 mg/mL.
- Precipitation: Precipitation was observed in the cytotoxicity Range-Finder experiment, as well as both experiments 1 and 2. No precipitate was observed at the time of treatment but following the 3 hour incubation period, precipitate was observed at the highest three concentrations in the absence and presence of S9 in the range-finder experiment. The highest concentration analysed was 500 ug/mL, which gave 87% and 91% RS with and without S9, respectively. In experment 1, precipitate was observed at the highest five concentrations tested in the absence of S9 (300 to 2000 ug/mL) and at the highest threee concentrations in the presence of S9 (500 to 2000 ug/mL). In experiment 2, precipitate was observed at the highest four concentrations in the absence and presence of S9 (400 to 2000 ug/mL). The lowest concentrations at which precipitates were observed at the end of the treatment incubation period, with and without S9, were retained and higher concentrations were discarded for all three experiments.

MUTAGENICITY (INCLUDING COMPARISON WITH HISTORICAL CONTROL DATA): In experiments 1 and 2, when tested up to precipitating concentrations, no statistically significant increases in MF were observed following treatment with ruthenium trichloride hydrate at any concentration analysed in the absence and presence of S9. A weakly statistically significant linear trend (p<0.05) was seen in the absence of S9 in experiment 2. However, as there were no statistically significant increases in MF at any concentration analysed and the effect was not reproduced between experiments, this isolated observation 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 S9) and B[a]P (with S9). Therefore the study was accepted as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The highest concentrations analysed in experiment 1 (300 and 500 μg/mL without and with S9 respectively) gave 107 and 72% RS respectively. The highest concentrations analysed in experiment 2 (400 μg/mL, - and + S9) gave 103 and 86% RS respectively.

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, conducted to GLP, ruthenium trichloride hydrate failed to induce biologically relevant increases in mutations at the hprt locus of L5178Y mouse lymphoma cells when tested up to precipitating concentrations in two independent experiments, each 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), ruthenium trichloride hydrate 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 500 μg/mL and above. In the main test, cells were exposed to test material for 3 hours in two independent experiments, each in the absence and presence of S9. Concentrations of 12.5-300 μg/mL and 25-500 μg/mL (Experiments 1 and 2, respectively) were used. The highest concentrations analysed, limited by precipitation, were 300 and 500 μg/mL (Experiment 1, without and with S9, respectively) exhibiting 107% and 72% relative survival (RS), respectively. In Experiment 2, the highest concentrations analysed was 400μg/mL, both without and with S9, which gave RS values of 103% and 86%, respectively.

No significant increases in mutant frequency (MF) over the concurrent controls were observed following treatment with ruthenium (III) chloride hydrate 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 weakly statistically significant linear trend (p<0.05) in MF was observed in the absence of S9 in Experiment 2, this was considered not biologically relevant since there were no statistically significant increases in MF at any concentration analysed and the effect was not reproduced between experiments.

Overall, ruthenium trichloride hydrate 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:

16 September - 9 November 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 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

no

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

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:

Cytotoxicity Range-Finder Experiment, 3+21 -S9, 3+21 +S9, 24+0 -S9: 7.256 to 2000 μg/mL
Micronucleus Experiment, 3+21 -S9, 3+21 +S9: 10 to 1000 μg/mL
Micronucleus Experiment, 24+0 -S9: 30 to 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:

clastogenicity

Positive control substance:

mitomycin C

Remarks:

3+21 -S9: 0.3 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

clastogenicity

Positive control substance:

cyclophosphamide

Remarks:

3+21 +S9: 2 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

aneugenicity

Positive control substance:

other: noscapine

Remarks:

24+0 -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 0 hours, 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: (micronucleus experiment) 500/culture (1000/concentration); except vehicle control, 500/culture (total 2000)

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); cytotoxicity is expressed as (100 - relative RI)

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 observed
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 observed by eye at the end of the treatment period

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 in each replicate were used to establish acceptable heterogeneity betweenreplicates by means of a binomial dispersion 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)
The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardsonet al. (1989). 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 peripheral blood lymphocytes

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: stock formulations: range-finder experiment, 20 mg/mL, pH 1.23; micronucleus experiment, 10 mg/mL, pH 1.54. A decrease in pH of >1 unit, compared to the concurrent vehicle control values, was observed at 2000 μg/mL under all three treatment conditions. However, the highest concentration tested in the micronucleus experiment was 1000 μg/mL and the change in pH at 1200 μg/mL was <1 unit under all treatment conditions, therefore the decrease in pH was not consdered by the investigators to be biologically relevant to the conduct of the study.
- 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. No further measurements of osmolality were made.
- Water solubility: soluble in purified water at a concentration of at least 56.15 mg/mL; the solubility limit in culture medium was<2807.5 μg/mL.
- Precipitation: visible precipitation at 2807.5 µg/mL which persisted for approximately 20 hours after test article addition. A maximum concentration of 2000 μg/mL was selected for the cytotoxicity range-finder experiment. Concentrations for the micronucleus experiment were based on the range-finder experiment. In the micronucleus test, precipitation was observed at the end of treatment incubation and/or at harvest at 200 μg/mL and above (3+21 hours +/-S9) or 60 μg/mL and above (24+0 hours -S9).

RANGE-FINDING/SCREENING STUDIES: cytotoxicity:
3+21 hours, -S9: up to 259.2 µg/mL, 0%; 432 µg/mL, 5%; 720 µg/mL, 13%, 1200 µg/mL, 39%; 2000 µg/mL, not evaluated (no scoreable cells).
3+21 hours, +S9: up to 432 µg/mL, 0%; 720 µg/mL, 1%; 1200 µg/mL, 17%; 2000 µg/mL, not evaluated (no scoreable cells).
24+0 hours, +S9: up to 33.59 µg/mL, 0%; 55.99 µg/mL, 2%; 93.31 µg/mL, 3%; 155.5 µg/mL, 0%; 259.2 µg/mL, 3%, 432 µg/mL, 5%; 720 µg/mL, 24%; 1200 µg/mL, 74%; 2000 µg/mL, not evaluated (no scoreable cells).

COMPARISON WITH HISTORICAL CONTROL DATA: frequency of micronucleated binucleate (MNBN) cells/cells scored(%):
Vehicle control
3+21 hours, -S9: mean, 0.46, 95% reference range, 0.10-1.30
3+21 hours, +S9: mean, 0.40, 95% reference range, 0.10-0.90
24+0 hours, -S9: mean, 0.52, 95% reference range, 0.10-1.50
Positive control
no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: micronucleus experiment:
3+21 hours, -S9: 125 µg/mL, 0%; 150 µg/mL, 1%, 200 µg/mL, 5%.
3+21 hours, +S9: 125 µg/mL, 2%; 150-200 µg/mL, 0%.
24+0 hours, -S9: 30 µg/mL, 1%; 60-90 µg/mL, 0%.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In a guideline in vitro micronucleus assay, conducted to GLP, ruthenium trichloride hydrate 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.

Executive summary:

Ruthenium trichloride hydrate 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 1000 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. The concentrations selected for analysis were 125, 150 and 200 μg/mL (3-hour treatment) and 30, 60 and 90 μg/mL (24-hour treatment).

There was no evidence of an increase in micronucleus frequency with or without S9 following treatment of cells with ruthenium trichloride hydrate for 3 hours and/or 24 hours. Some evidence of cytotoxicity was observed at 200 µg/mL though this was not statistically significant. Vehicle and positive controls performed as expected.

Overall, ruthenium trichloride hydrate 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

Ruthenium trichloride hydrate 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). 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. In experiment 1 a plate incorporation protocol was used; the experiment was repeated using a pre-incubation step. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition. There was no evidence of mutagenicity in any strain with or without S9 in either experiment. Some evidence of toxicity was observed at 1600 and/or 5000 µg/plate with the plate incorporation protocol and at 1250 and/or 2500 µg/plate with the pre-incubation protocol. Vehicle and positive controls performed as expected. It is concluded that ruthenium trichloride hydrate 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), ruthenium trichloride hydrate 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 500 μg/mL and above. In the main test, cells were exposed to test material for 3 hours in two independent experiments, each in the absence and presence of S9. Concentrations of 12.5-300 μg/mL and 25-500 μg/mL (Experiments 1 and 2, respectively) were used. The highest concentrations analysed, limited by precipitation, were 300 and 500 μg/mL (Experiment 1, without and with S9, respectively) exhibiting 107% and 72% relative survival (RS), respectively. In Experiment 2, the highest concentrations analysed was 400 μg/mL, both without and with S9, which gave RS values of 103% and 86%, respectively. No significant increases in mutant frequency (MF) over the concurrent controls were observed following treatment with ruthenium (III) chloride hydrate 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 weakly statistically significant linear trend (p<0.05) in MF was observed in the absence of S9 in Experiment 2, this was considered not biologically relevant since there were no statistically significant increases in MF at any concentration analysed and the effect was not reproduced between experiments. Overall, ruthenium trichloride hydrate 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).

 

Ruthenium trichloride hydrate 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 1000 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. The concentrations selected for analysis were 125, 150 and 200 μg/mL (3-hour treatment) and 30, 60 and 90 μg/mL (24-hour treatment). There was no evidence of an increase in micronucleus frequency with or without S9 following treatment of cells with ruthenium trichloride hydrate for 3 hours and/or 24 hours. Some evidence of cytotoxicity was observed at 200 µg/mL though this was not statistically significant. Vehicle and positive controls performed as expected. Overall, ruthenium trichloride hydrate 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, ruthenium trichloride hydrate does not meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008).