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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, diammonium sodium hexakis(nitrito-N)rhodate 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, 2014).

 

In an in vitro mammalian cell gene mutation assay, conducted in accordance with OECD Test Guideline 476 and to GLP, diammonium sodium hexakis(nitrito-N)rhodate did not induce biologically relevant increases in mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to precipitating concentrations in the absence and presence of S9 (Lloyd, 2014a).

 

In an in vitro micronucleus assay, conducted according to OECD Test Guideline 487 and to GLP, diammonium sodium hexakis(nitrito-N)rhodate failed to induce biologically relevant increases in micronuclei in Chinese hamster ovary cells when tested up to the limit of cytotoxicity, for 3 hours in the absence and presence of S9, and for 24 hours without S9 (Lloyd, 2014b).

Link to relevant study records
Reference
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
Qualifier:
according to
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
Target gene:
Histidine
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
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
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
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.

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

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 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. 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 (McGarry, 2014).

 

In an in vitro GLP study, conducted in accordance with OECD Test Guideline 476 (in vitro mammalian cell gene mutation assay), diammonium sodium hexakis(nitrito-N)rhodate 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 (46.88-1500 μg/mL) were tested (with and without S9); precipitation was seen after 3 hours incubation at the two highest concentrations, with and without S9. The main test consisted of two experiments, each using an incubation time of 3 hours and testing 10 concentrations with and without S9. Precipitation was observed at the end of the incubation period in both experiments: above 400 μg/mL (Experiment 1) and above 300 μg/mL (Experiment 2), with and without S9 in both cases. In Experiment 1, small but statistically significant increases in mean mutant frequency (MF) over the concurrent vehicle control value were observed at two intermediate concentrations (250 and 350 μg/mL) in the absence of S9. However, these values were less than three times the historical mean vehicle control value and this effect was not observed at higher doses (there was no statistically significant linear trend). Moreover, the increases were not reproducible as in the absence of S9 in Experiment 2. Therefore, the small, sporadic increases in MF seen in the absence of S9 in Experiment 1 were considered of little or no biological relevance. In the presence of S9 when tested up to precipitating concentrations, no statistically significant increases in mean MF were observed at any concentration analysed in Experiments 1 and 2 and there were no significant linear trends. Overall, diammonium sodium hexakis(nitrito-N)rhodate did not induce biologically relevant increases in gene mutations at the hprt locus of L5178Y mouse lymphoma cells when tested up to cytotoxic concentrations in two independent experiments in the absence and presence of S9 (Lloyd, 2014a).

 

Diammonium sodium hexakis(nitrito-N)rhodate was tested for its ability to induce chromosome damage (micronuclei) in Chinese hamster ovary cells in an assay conducted in accordance with OECD Test Guideline 487 and to GLP. The highest concentrations of test article analysed were limited by toxicity and were determined following a preliminary cytotoxicity range-finder experiment. Cells were treated with the test material (in 1% methyl cellulose) for either 3 hours (with 21 hours recovery time), or for 24 hours, up to cytotoxic concentrations in the absence (24-hour treatments) or presence and absence (3-hour treatments) of S9. Appropriate vehicle and positive control cultures were included in the test system under each treatment condition and matched the acceptance criteria. Treatment of cells with diammonium sodium hexakis(nitrito-N)rhodate for 3 hours and for 24 hours in the absence of S9 resulted in frequencies of micronucleated binucleate MNBN cells that were generally similar to (marginally but not significantly higher than) those observed in concurrent vehicle controls at the various concentrations analysed under both treatment conditions. A statistically significant increase in micronuclei was observed at the highest concentration (1300 μg/mL) in the 3-hour treatment in the absence of S9, but the effect was small and the frequency of micronucleated cells was well within the historical control range. A statistically significant increase in micronuclei was also observed at the highest-tested concentration (400 μg/mL) in one of two experimental runs of the 24-hour treatment (without S9), but this effect was found to be not reproducible in a similar experiment (also conducted in duplicate but up to a slightly less cytotoxic concentration). As a result, neither of these changes was considered biologically relevant. Overall, diammonium sodium hexakis(nitrito-N)rhodate did not induce biologically relevant increases in the frequency of micronuclei in Chinese hamster ovary cells treated in culture, when tested up to the limit of cytotoxicity in the presence and absence (3-hour treatment) or absence (24-hour treatment) of S9 (Lloyd, 2014b).

Justification for classification or non-classification

Based on the existing data set, diammonium sodium hexakis(nitrito-N)rhodate does not currently meet the criteria for classification as a germ cell mutagen (category 1A or 1B) under EU CLP criteria (EC 1272/2008). However, in spite of the negative results following in vitro genotoxicity testing, based on the anticipated speciation of diammonium sodium hexakis(nitrito-N)rhodate in vivo (dissociation and binding with chloride ligands in the gastric environment), the substance is classified as a category 2 mutagen on a precautionary basis.