Dirhodium trisulphate

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:

4 November - 16 December 2014 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Rhodium sulphate

IUPAC Name:

Rhodium sulphate

Test material form:

other: lquid

Details on test material:

- Name of test material (as cited in study report): Rhodium sulphate
- Substance type: No data
- Physical state: Brown liquid
- Analytical purity: No data
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: Rhodium sulphate solution type R100S (8.13% w/w rhodium content)
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: GY0072
- Expiration date of the lot/batch: 21 May 2017
- Stability under test conditions: No data
- Storage condition of test material: 15-25 deg C, protected from light
- Other: Purity was stated as 8.13% based on rhodium content - assumed to be 100% for testing

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

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:

Range-finder experiment and Experiment 1: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate (both with and without S9)
Experiment 2: 160, 300, 625, 1250, 2500 and 5000 μg/plate (both with and without S9)

NB. It is unclear whether these concentrations relate to amounts rhodium sulphate itself or amounts of rhodium sulphate solution (in which case the actual rhodium sulphate concentration would be less than stated above).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Test guideline recommends use of aqueous solvent wherever possible

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

5 μg/plate for TA 98 without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 μg/plate for TA 100 and TA 1535 without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 μg/plate for TA 1537 without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.2 μg/plate for TA 102 without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

10 μg/plate for TA98 with S9

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

5 μg/plate for TA 100, TA 1535 and TA 1537 with S9; 20 μg/plate for TA 102 with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). Platings were achieved by the following sequence of additions to 2.5 mL of 0.9% molten agar at 46±1 deg C: 0.1 mL bacterial culture, 0.1 mL test article solution or control, 0.5 mL 10% S-9 mix or buffer solution. This was followed by rapid mixing and pouring on to Vogel-Bonner E agar plates.
A pre-incubation step was included for experiment 2 in the presence of S9. Quantities of test article or control solution (reduced to 0.05 mL for the positive control treatments), bacteria and S9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1 deg C, with shaking, before the addition of 2.5 mL molten agar at 46±1 deg C.

DURATION
- Preincubation period: 20 minutes (experiment 2 only)
- Exposure duration: 3 days
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: The mutation experiments were performed in triplicate

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Examination of the background lawn for signs of toxicity or a reduction in number of revertant colonies/reduction in mutagenic response compared to vehicle controls

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

Evaluation criteria:

The test article was considered to be mutagenic if a) a concentration related increase in revertant numbers was >=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 values; b) the positive trend/effects described above were reproducible.

The test article was considered positive if both criteria were met and negative if neither criteria were met.

Statistics:

Not applicable

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(In the absence and presence of S9 in both experiments)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(The test material caused a slight thinning of the bacterial lawn at 5000 μg/plate in experiment 2 in the absence and presence of S9. Evidence of cytotoxicity was observed in experiment 1 at 500 and 5000 μg/plate in the absence of S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(In the absence and presence of S9 in both experiments)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(The test material caused a slight thinning of the bacterial lawn at 5000 μg/plate in experiment 2 in the absence and presence of S9. No evidence of cytotoxicity was observed in experiment 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(At 5000 μg/plate in the presence of S9 in experiment 2, with additional evidence of mutagenicity in the absence of S9 in both experiments 1 and 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(The test material caused a slight thinning of the bacterial lawn at 5000 μg/plate in experiment 2 in the absence and presence of S9. No evidence of cytotoxicity was observed in experiment 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(At 5000 μg/plate in the absence of S9 in experiment 1 and at 5000 and 1250-5000 μg/plate in the absence and presence of S9, respectively, in experiment 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(The test material caused a slight thinning of the bacterial lawn at 5000 μg/plate in experiment 2 in the absence and presence of S9. No evidence of cytotoxicity was observed in experiment 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(At 5000 μg/plate in the absence and presence of S9 in experiment 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(The test material caused a slight thinning of the bacterial lawn at 5000 μg/plate in experiment 2 in the absence and presence of S9. No evidence of cytotoxicity was observed in experiment 1)

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: The pH measurements of the stock formulations prior to treatment were 1.23, 1.79 and 1.80 for the range-finder experiment, experiment 1 and experiment 2, respectively.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: Preliminary solubility data indicated that Rhodium Sulfate was soluble in water for irrigation (purified water) at concentrations of at least 50 mg/mL.
- Precipitation: The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The concentration range used was 0, 5, 16, 50, 160, 500, 1600 and 5000 μg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: The mean vehicle control counts fell within the laboratory’s historical ranges. The positive control chemicals all induced increases in revertant numbers of >=1.5-fold (in strain TA 102), >=2-fold (in strains TA 98 and TA 100) or >=3-fold (in strains TA 1535 and TA 1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finder experiment 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.

Remarks on result:

other: strain/cell type: TA 1537

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Increases in revertants approaching the 2-fold threshold (1.8‑fold the concurrent vehicle controls) were also observed in strain TA 100 in the absence of S9 in both Experiment 1 and Experiment 2. As these increases in revertants were very close to the 2-fold threshold on more than one experimental occasion, these increases are considered as possible further evidence of mutagenic activity in this strain.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

In a guideline study, to GLP, rhodium sulphate was found to be mutagenic in Salmonella typhimurium strains TA 98, TA 100 and TA 102 when tested in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

In an OECD Test Guideline 471 study, conducted according to GLP, rhodium sulphate was examined for the ability to induce gene mutations in Salmonella typhimurium (strains TA 98, TA 100, TA 102, TA 1535 and TA 1537) in the presence and absence of Aroclor-induced rat liver metabolic activation system (S9). Two experiments (each in triplicate) were conducted for each strain using the plate incorporation method, with both involving concentrations of up to 5000 µg/plate, based on observed precipitation in a range-finding study. The second experiment was conducted with an additional pre-incubation step.

 

In experiment 1, no significant increases in mutant revertants were observed in any strain in the presence of S9. In the absence of S9, strain TA 102 showed a significant increase in revertants (with no associated cytotoxicity).

 

In experiment 2, strains TA 98 and TA 102 (both in the absence and presence of S9) and TA 100 (in the presence of S9) showed significant increases in revertants, though for the most part this was at the cytotoxic level of 5000 μg/plate. A significant increase in revertant numbers at concentrations below the cytotoxic level was observed in strain TA 102 in the presence of S9. No significant increases in revertant numbers were seen in strains TA 1535 and TA1537.

 

Overall, rhodium sulphate was found to be mutagenic in S. typhimurium strains TA 98, TA 100 and TA 102 in the absence and presence of S9 under the conditions of the test.

 

[It is unclear whether the tested concentrations relate to amounts rhodium sulphate itself or amounts of rhodium sulphate solution].