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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 March 2018 - 9 April 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Ruthenium dioxide (hydrate)
Cas Number:
Ruthenium dioxide (hydrate)
Constituent 2
Chemical structure
Reference substance name:
Ruthenium (IV) oxide
EC Number:
EC Name:
Ruthenium (IV) oxide
Cas Number:
Molecular formula:
ruthenium (IV) oxide
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Ruthenium(IV)oxid hydrat
- Physical state: Powder
Specific details on test material used for the study:
Purity: 78.08% Ru content


Target gene:
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S-9 derived from Aroclor 1254-treated male Sprague-Dawley rats
Test concentrations with justification for top dose:
Mutation Experiment 1 (all strains; with and without S9)
5, 16, 50, 160, 500, 1600, 5000 ug/plate

Mutation Experiment 2 (all strains; with and without S9)
20.48, 51.2, 128, 320, 800, 2000, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test article stock formulations were prepared by suspending Ruthenium(IV) oxide
under subdued lighting in DMF with the aid of vortex mixing, ultrasonication and
warming at 37°C, to give the maximum required treatment concentration. Subsequent
dilutions were made using DMF. All test article formulations and dilutions were
treated as though they were suspensions, and mixed (by inversion or agitation)
immediately prior to each dilution or treatment.

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Ruthenium(IV) oxide was insoluble in
several vehicles commonly used in this assay system, including tetrahydrofuran
(THF) ethanol and dimethylformamide (DMF). The test article did however provide a
homogenous and stable suspension when formulated in DMF at 100 mg/mL, and
formulation in this vehicle was used in this study.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
sodium azide
mitomycin C
other: 2-aminoanthracene
2NF for TA98 (-S9); NaN3 for TA100 and TA1535 (-S9); AAC for TA1537 (-S9); MMC for TA102 (-S9); BaP for TA98 (+S9); AAN for TA100, TA1535, TA1537 and TA102 (+S9)
Details on test system and experimental conditions:
- In agar (plate incorporation); preincubation (for experiment 2 in the presence of S9).
- 0.05 mL volume additions of test article solution were used for all experiments

Triplicate plates for test substance, vehicle and positive controls.

Prepared test suspensions were protected from light and used within approximately 7.5 hours of initial formulation.

As the results of Experiment 1 were negative, treatments in the presence of S-9 in
Experiment 2 included a pre-incubation step. Quantities of test article, vehicle,
untreated or positive control, bacteria and S-9 mix detailed above, were mixed
together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C.

Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity included a marked reduction in revertants compared to the concurrent vehicle controls.

Evaluation criteria:
Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.

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 > (or equal to) 1.5-fold (in strain TA102), > (or equal to) 2-fold (in strains TA98 and TA100) or > (or equal to) 3-fold (in strains TA1535 and TA1537) the concurrent vehicle control, confirming discrimination between different strains, and an active S 9 preparation.

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. Any observed response was reproducible under the same treatment conditions.
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 either of the above criteria were met.

Individual plate counts were recorded separately and the mean and standard deviation
of the plate counts for each treatment were determined. Control counts were
compared with the laboratory’s historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical
analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).
However, adequate interpretation of biological relevance was of critical importance.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Exp1: evidence of toxicity at 5000 µg/plate (TA98,100,1537 and 102 -S-9; TA98 and 102 + S-9) Exp2: evidence of toxicity at 5000 µg/plate (TA1535 and 102 -S-9; TA100,1537 and 102 + S-9)
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
pH assessments were performed on all the test article treatment concentrations in each
experiment. It may be noted that due to the apparent variability of the pH values, and lack of any clear
pattern of increase or decrease across the concentration range, the pH assessments on
the samples of the treatment suspension concentration in Mutation Experiment 1 were
repeated. The pH values in Mutation Experiment 1 mostly fell within or close to one
pH unit across the concentration range, with the exception of the lowest (0.1 mg/mL)
formulation concentration, which had a pH value somewhat lower than that of the
other concentrations. In Mutation Experiment 2, most of the treatment formulation
concentrations provided pH values within one pH unit, the exception being the
highest treatment suspension concentration (100 mg/mL) which had a slightly lower
pH value, and the 1.024 mg/mL formulation which had a slightly higher pH value. In
each experiment, as the majority of treatment concentrations fell with on close to one
pH unit of each other, they were considered acceptable.

An intense colouration was noted on all plates treated at 2000 μg/plate and above in
each experiment.

The vehicle control counts were within the laboratory’s historical ranges, with the exception of a
single vehicle control replicate plate count in strain TA1537 in the presence of S-9 in
Mutation Experiment 1, which fell slightly below the laboratory historical control
range. This count was however sufficiently comparable to the other replicate counts
and to the historical range to be considered characteristic and acceptable.

Applicant's summary and conclusion

In a guideline Ames test, it was concluded that Ruthenium(IV) oxide did not induce mutation in fivehistidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. Theseconditions included treatments at concentrations up to 5000 μg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

Ruthenium(IV) oxide was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Ruthenium(IV) oxide formulations in this study were prepared as suspensions in dimethylformamide (DMF), and all treatments were performed using these formulations and subsequent dilutions as though they were suspensions.

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Ruthenium(IV) oxide at 5-5000 μg/plate. Following these treatments, evidence of toxicity was observed at 5000 μg/plate in most strains in the absence and/or presence of S-9. Although treated as suspensions, this toxicity confirmed

exposure of the test article to the assay system.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 20.48-5000 μg/plate. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a

pre-incubation step. Following these treatments, evidence of toxicity was again observed in most strains in the absence and/or presence of S-9.

pH assessments were performed on all the test article treatment concentrations in each experiment. In Mutation Experiment 1, the pH values ranged from 8.11 to 10.28 for the initial assessments and 7.69 to 9.06 for the repeat assessments. In Mutation Experiment 2 the pH values ranged from 10.75 to 12.41.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Ruthenium(IV) oxide treatments of all the test strains in the absence and presence of S-9, no notable and concentration-related increases in revertant numbers were observed, and none that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Ruthenium(IV) oxide mutagenic activity in this assay system.

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