Diammonium sodium hexakis(nitrito-N)rhodate

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:

08 November 2013 - 10 February 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-conducted study performed in accordance with OECD guidelines (with acceptable minor deviations) and to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Vehicle revertant count (mean and one individual count in strain TA102, expt 2) with S9 was outside 99% CI. Marginal difference. Data considered valid. Not considered to affect overall interpretation of study findings or study integrity.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium sodium hexakis(nitrito-N)rhodate

EC Number:

264-713-0

EC Name:

Diammonium sodium hexakis(nitrito-N)rhodate

Cas Number:

64164-17-6

Molecular formula:

H4N.1/2N6O12Rh.1/2Na

IUPAC Name:

Diammonium sodium hexakis(nitro-N)rhodate

Constituent 2

Reference substance name:

Rhodium nitrite salt

IUPAC Name:

Rhodium nitrite salt

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Diammonium sodium hexakis(nitrito-N)rhodate
- Substance type: technical product
- Physical state: solid (green powder)
- Analytical purity: assumed 100% for experimental suspension formulation
- Impurities (identity and concentrations): traces of platinum (0.01%), ruthenium (0.073%), iridium (0.044%), silver (0.016%); traces of other metals (iron, nickel, bismuth, copper, tellurium, chromium, magnesium) documented in the study report's certificate of analysis
- Composition of test material, percentage of components: Rhodium 16.63%; sodium 3.88%
- Purity test date: 01 April 2014
- Lot/batch No.:RHL 653
- Expiration date of the lot/batch: 03 June 2015. The expiry date of the test article was based on two years from the production date of the material. As far as the manufacturer is aware, no degradation/aging is expected in any long term time frame however there is no analytical data available to confirm this.
- Stability under test conditions: test article stability in the vehicle was not determined in this study; fresh preparations were routinely employed.
- Storage condition of test material: 15-25 degrees C, protected from light

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)
160
300
625
1250
2500
5000 ug/plate

Mutation Experiment 2 Additional work in strain TA100 (without S9)
500
1000
2000
3000
4000
5000 ug/plate

Mutation Experiment 2 Additional work in strain TA1537 (without S9)
100
250
750
1000
1250
2500
5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: test material was applied in a homogenous suspension: approximately 10.00 mg/mL in 1% methylcellulose (1% MC (high viscosity)). To ensure homogeneity was maintained throughout treatment each formulation was stirred continuously using a magnetic stirrer.
- Justification for choice of solvent/vehicle: the test material was insoluble in several commonly used solvents including dimethylformamide (DMF), ethanol, water for irrigation (purified water), tetrahydrofuran (THF), acetone and acetonitrile

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

As 1% MC (high viscosity) is not commonly used as a vehicle in this laboratory, untreated control treatments (UTC) comprising 100 mM sodium phosphate buffer (pH 7.4) were also included

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).
- 0.5 mL volume additions of test article solution were used for all treatments (except experiment 2 pre-incubation treatments for positive control): 0.05 mL due to the vehicle (DMSO) employed.

Triplicate plates for test substance and positive controls. Vehicle and untreated controls were tested in quintuplicate.

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 or control solution, bacteria and S 9 mix, were mixed together and incubated for 20 minutes at 37±1 degreesC, with shaking, before the addition of 2.5 mL molten agar at 46±1 degrees C.

DETERMINATION OF CYTOTOXICITY
Initial range-finder experiment was carried out in strains TA98, TA100 and TA102 (with and without S9), plus vehicle, untreated and positive controls.There was no clear evidence of toxicity up to a test-material concentration of 5 mg/plate.

The background lawns of all plates were examined for signs of toxicity.

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.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined.

The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels

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

Genotoxicity:

negative

Remarks:

Expt 2 treatment in TA1537 produced a notable increase in revertants at the highest non-precipitating concentration. This was considered not to be a mutagenic effect (see below).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Or a maximum test concentration of 5 mg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 2 treatments in strain TA1537 (without S9) at 1250 ug/plate (the highest non-precipitating concentration) resulted in an increase in revertant numbers (≥3-fold the concurrent control). The increase was small in magnitude and there was no clear indication of a concentration relationship or reproducibility between experiments; it was considered to have been due to normal biological variability and not evidence of mutagenic activity.
It should also be noted that following Experiment 2 treatments in strain TA100 in the absence of S 9, some of the untreated control revertant colony counts were higher than the historical control ranges and increases of 1.6-fold the concurrent vehicle controls were observed. In order to confirm if these increases were due to normal biological variability or mutagenicity, Experiment 2 additional treatments were performed in this strain. Following these treatments, no increases were observed that were ≥2-fold the concurrent vehicle controls.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

In a guideline Ames test, diammonium sodium hexakis(nitrito-N)rhodate failed to induce an increase in mutation frequency in five 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.

Executive summary:

Diammonium sodium hexakis(nitrito-N)rhodate 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 Salmonella typhimurium, both in the absence and in the presence of metabolic activation (S9), in two separate experiments. 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 2, treatments in the presence of S9 included a pre-incubation step. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and fit the acceptance criteria.

 

Following treatments of all the test strains in the absence and presence of S9, only Experiment 2 treatments in strain TA1537 in the absence of S9 at 1250 μg/plate (the highest non-precipitating concentration) resulted in an increase in revertant numbers that was ≥3-fold the concurrent control. The increase was small in magnitude and occurred at a single intermediate concentration with no clear indication of a concentration relationship or reproducibility between experiments. Accordingly this was not considered evidence of mutagenic activity. Following Experiment 2 treatments in strain TA100 in the absence of S9, some of the untreated control revertant colony counts were higher than the historical control ranges and increases of 1.6-fold the concurrent vehicle controls were observed. In order to confirm if these increases were due to normal biological variability or mutagenicity, Experiment 2 additional treatments were performed in this strain; no increases were observed that were ≥2-fold the concurrent vehicle controls.

 

It is concluded that diammonium sodium hexakis(nitrito-N)rhodate 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.