Dirhodium trioxide

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:

22 August 2016 - 16 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Dirhodium trioxide

EC Number:

234-846-9

EC Name:

Dirhodium trioxide

Cas Number:

12036-35-0

Molecular formula:

Rh2O3

IUPAC Name:

Rhodium(III)oxide

Details on test material:

Dirhodium trisoxide

Specific details on test material used for the study:

Purity: 81.08% Rhodium metal (equivalent to 100% Rhodium (III) oxide)

Method

Target gene:

Histidine

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:

Range-Finder Experiment and Mutation Experiment 1 (with and without S9)
5, 16, 50,160, 500, 1600, 5000 ug/plate

Mutation Experiment 2 (with and without S9)
51.2, 128, 320, 800, 2000, 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: the test article formed a homogenous suspension at 50 mg/mL in 0.5% (w/v) methyl cellulose (400 CPS) (0.5% MC). Test article stock suspensions were prepared by suspending Rhodium (III) oxide in 0.5% MC under subdued lighting and with the aid of Silverson mixing and stirring to give the maximum required treatment concentration. Subsequent dilutions were made using 0.5% MC. All test article formulations were stirred continuously throughout dilution and treatment to ensure homogeneity.
- Justification for choice of solvent/vehicle:Preliminary solubility assessments indicated that Rhodium (III) oxide was insoluble in several vehicles compatible with the assay system, including water, ethanol, acetone, dimethylformamide (DMF) and tetrahydrofuran (THF)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

untreated controls comprised treatments with either 100 mM sodium phosphate buffer (in the Range-Finder Experiment) or purified water (in Mutation Experiments 1 and 2)

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Remarks:

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:

METHOD OF APPLICATION:
- In agar (plate incorporation); preincubation (for experiment 2 in the presence of S9).
- 0.1 mL volume additions of test article suspension were used for all treatments (positive controls were treated using 0.05 mL volume additions)/

Triplicate plates for test substance and positive controls. Vehicle and untreated controls were tested in quintuplicate (triplicate for experiment 2).

Prepared test suspensions were protected from light and used within approximately 3 hours of initial formulation.

DURATION
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 (see Protocol Deviations, Section 7.5) 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.

DETERMINATION OF CYTOTOXICITY
The test item was tested for toxicity (and mutation) in strain TA98, TA100 and
TA102. Duplicate plates without and with
S- 9 mix were used for vehicle, untreated and positive controls; single plates were
used for the test article.

These platings were achieved by the following sequence of
additions to 2 mL of molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article suspension, vehicle, untreated or positive control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to agar plates. When set,
the plates were inverted and incubated at 37±1°C protected from light for 3 days.

Following incubation, these plates were examined for evidence of toxicity to the
background lawn and, where possible, revertant colonies were counted.

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.

Statistics:

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

Test results 1

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In a guideline Ames test, it was concluded that Rhodium (III) oxide did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested at concentrations up to 5000 μg/plate for each strain, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Rhodium (III) 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. As no vehicle could be identified that would dissolve the test article and was compatible with the assay system, all Rhodium (III) oxide treatments in this study were performed using suspensions prepared in 0.5% Methyl Cellulose (0.5% MC).

An initial toxicity Range-Finder Experiment was carried out in the absence and in the presence of S-9 in strains TA98, TA100 and TA102 only, using final concentrations of Rhodium (III) oxide up to 5000 μg/plate. No evidence of toxicity was observed.

The same concentrations were retained for treatments of all the tester strains in Experiment 1 in the absence and in the presence of S-9. Following these treatments, no evidence of toxicity was observed.

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 ranges 51.2 - 5000 μg/plate. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a preincubation step. Following these treatments, no clear evidence of toxicity was observed.

The test article was treated as a suspension in this study, and therefore any observations of precipitation are not considered relevant and are not reported.

Vehicle, untreated and positive control treatments were included for all strains in all experiments. The numbers of revertant colonies on vehicle control plates were comparable with acceptable ranges and with the untreated controls, and were elevated by positive control treatments.

Following Rhodium (III) oxide treatments of all the test strains in the absence and presence of S-9 in the mutation experiments, no notable or 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 Rhodium (III) oxide mutagenic activity in this assay system.